Transdermal assay with magnetic clamp

ABSTRACT

The transdermal assay apparatus includes first, second, and third members. The first member has one or more sample surfaces, each of which is configured to receive a sample thereon. The second member defines one or more reservoirs, each of which has an opening on a surface of the second member. Each sample surface is substantially the same size as each opening. The transdermal assay apparatus also includes a magnetic clamp configured to clamp a tissue specimen between the sample surface and the opening. The magnetic clamp preferably includes a magnet having a strength that is selected based on the clamping force required between the first member and the second member. The invention also provides a method for using a transdermal assay apparatus.

[0001] This application is a divisional of U.S. application Ser. No.10/282,505, filed Oct. 28, 2002, which is a continuation-in-part of U.S.application Ser. No. 09/904,725, filed Jul. 13, 2001, which claims thebenefit of provisional application No. 60/240,891, filed Oct. 16, 2000;provisional application No. 60/220,324, filed Jul. 24, 2000; provisionalapplication No. 60/218,377, filed Jul. 14, 2000, each of which is herebyincorporated by reference.

1. FIELD OF THE INVENTION

[0002] The field of the present invention relates to tissue barrierassays for screening formulations and chemical compositions.

2. BACKGROUND OF THE INVENTION

[0003] In vitro analysis of the movement of compounds (e.g., drugs)across an epithelial barrier, such as intestinal epithelium or airwayepithelium, is typically performed using an Ussing-type chamber. Toperform a tissue barrier assay using an Ussing-type chamber, a piecetissue is removed as an intact sheet from the body and mounted in adevice which contains an enclosed, internal hollow chamber such that itdivides the internal chamber into two separate chambers. Thereafter,biologically compatible solutions are filled into both chambers, and thedrug of interest is added to one chamber's solution. Samples are thenremoved from the contralateral chamber solution at various times todetermine the rate at which the drug moves across the tissue barrier.This type of tissue barrier assay is cumbersome, inefficient, and onlypermits a very limited number of independent samples to be derived froma unit area of tissue sheet.

[0004] Transdermal delivery of drugs is a type of tissue transfer thatinvolves transfer of the drug from a transdermal drug delivery devicethrough the skin and into the patient's blood stream. Transdermal drugdelivery offers many advantages compared to other methods of drugdelivery. One obvious advantage is that needles and the associated painare avoided. This is especially desirable for drugs that are repeatedlyadministered. Avoiding the unpleasantness of needles would also lead toimproved patient compliance of drug regimens.

[0005] Another advantage of transdermal drug delivery is its ability tooffer prolonged or sustained delivery, potentially over several days toweeks. Other delivery methods, such as oral or pulmonary delivery,typically require that the drug be given repeatedly to sustain theproper concentration of drug within the body. With sustained transdermaldelivery, dose maintenance is performed automatically over a long periodof time. This is especially beneficial for drugs with short half-livesin the body, such as peptides or proteins.

[0006] A final advantage is that drug molecules only have to cross theskin to reach the bloodstream when given transdermally. Transdermallyadministered drugs bypass first-pass metabolism in the liver, and alsoavoid other degradation pathways such as the low pH's and enzymespresent in the gastrointestinal tract.

[0007] The skin is the largest organ of the body. It is highlyimpermeable to prevent loss of water and electrolytes. It is subdividedinto two main layers: the outer epidermis and the inner dermis. Theepidermis is the outer layer of the skin, 50 to 100 micron thick(Monteiro-Riviere, 1991; Champion, et al., 1992). The dermis is theinner layer of the skin and varies from 1 to 3 mm in thickness. The goalof transdermal drug delivery is to get the drug to this layer of theskin, where the blood capillaries are located, to allow the drug to besystemically delivered. The epidermis does not contain nerve endings orblood vessels. The main purpose of the epidermis is to generate a toughlayer of dead cells on the surface of the skin, thereby protecting thebody from the environment. This outermost layer of epidermis is calledthe stratum corneum, and the dead cells that comprise it are calledcorneocytes or keratinocytes.

[0008] The stratum corneum is commonly modeled or described as a brickwall (Elias, 1983; Elite, 1988). The “bricks” are the flattened, deadcorneocytes. Typically, there are about 10 to 15 corneocytes stackedvertically across the stratum corneum (Monteiro-Riviere, 1991; Championet al., 1992). The corneocytes are encased in sheets of lipid bilayers(the “mortar”). The lipid bilayer sheets are separated by 50 nm.Typically, there are about 4 to 8 lipid bilayers between each pair ofcorneocytes. The lipid matrix is primarily composed of ceramides,sphingolipids, cholesterol, fatty acids, and sterols, with very littlewater present (Lampe et al., 1983 [a]; Lampe et al., 1983 [b]; Elias,1988).

[0009] Although it is the thinnest layer of the skin, the stratumcorneum is the primary barrier to the entry of molecules ormicroorganisms across the skin. Most molecules pass through the stratumcorneum only with great difficulty, which is why the transdermal drugdelivery route has not been more widely used to date. Once the moleculeshave crossed the stratum corneum, diffusion across the epidermis anddermis to the blood vessels occurs rapidly. Thus, most of the attentionin transdermal drug delivery research has been focused on transportingmolecules and drugs across the stratum corneum.

[0010] The most common form of transdermal drug delivery device is thetransdermal drug “patch,” where a drug, or pharmaceutical, is containedwithin a reservoir placed next to the skin (Schaefer and Redelmeier,1996). The drug molecules typically cross the skin by simple diffusion.Transport is governed by the rate of molecular diffusion into and out ofthe skin, and partitioning of the drug into the skin. Generallyspeaking, transdermal drug delivery is limited to small, lipophilicmolecules such as scopolamine, nitroglycerine, and nicotine, whichreadily permeate the skin. The delivery is slow, typically taking hoursfor the drug to cross the skin, and treatment is only effective when avery small amount of drug is required to have a biological effect (Guyand Hadgraft, 1989).

[0011] Since transdermal delivery can be slow, many substances have beenused to enhance molecular transport rates. These substances are known aschemical enhancers or penetration enhancers. Chemical enhancers increasethe flux of a drug through the skin by increasing the solubility of drugin the stratum corneum or increasing the permeability of drug in thestratum corneum. There are many possible enhancers and the selection isfurther complicated by the fact that combinations of enhancers are knownto improve drug flux beyond what would be expect due to the presence ofeach constituent independently.

[0012] Transdermal drug delivery devices, such as a transdermal patch,also generally contain an adhesive, which serves to keep the device inintimate contact with the skin, and may also form the matrix in whichthe drug is dissolved or dispersed. There are many different forms ofadhesives that can be used, and it is often a very difficult problem toselect which adhesive to use with any drug or drug and enhancer.

[0013] Currently, the choice of appropriate adhesive and enhancers andtheir relative proportion with respect to the drug is only determined bygeneral guidelines from what is known to be safe and what may have beeneffective with other drugs. The vast majority of the formulationdevelopment is made through trial and error experimentation.

[0014] Most transdermal transport experiments to date have utilized arelatively large human skin diffusion cell in which a source sideincludes a drug solution with additives and a sink side that typicallyincludes saline solution or some other solution that is thought to modelthe dermis. The skin membrane separates the two sides of the cell, andis most often stratum corneum cadaver skin that has been carefullyseparated from the whole skin sample supplied by a tissue bank. Thevolume of the device is typically 5 cc or greater. Samples areperiodically taken from the sink side of the cell to determine the fluxof drug through the stratum corneum film. The entire procedure is verylaborious and requires the use of large quantities of skin, which isextremely difficult to obtain. Therefore, only a relatively small numberof the many possible combinations of chemical entities can be examined.Also, only a limited number of formulations can be tested on a singledonor's skin, which makes it more difficult to compare the effect ofthose formulations, due to the additional variation in measurementintroduced by the response from different donor's skin samples.

[0015] Thus, there remains a need in the art for an apparatus and methodfor identifying optimal compositions or formulations for tissue barriertransport, including transdermal transport, of compounds,pharmaceuticals and other components. Such an apparatus should be easyto assemble. The apparatus should be sealable, wherein the requiredclamping force and sealing pressure between the drug or pharmaceutical,the skin membrane, and the saline or other solution used to model thedermis, is even and does not interfere with the function.

3. SUMMARY OF THE INVENTION

[0016] The present invention relates to high-throughput systems andmethods to prepare a large number of component combinations, at varyingconcentrations and identities, at the same time, and high-throughputmethods to test tissue barrier transfer of components in eachcombination. The methods of the present invention allow determination ofthe effects of additional or inactive components, such as excipients,carriers, enhancers, adhesives, and additives, on transfer of activecomponents, such as pharmaceuticals, across tissue, such as skin, lungtissue, tracheal tissue, nasal tissue, bladder tissue, placenta, vaginaltissue, rectal tissue, stomach tissue, gastrointestinal tissue, and eyeor corneal tissue. The invention thus encompasses the high-throughputtesting of pharmaceutical compositions or formulations in order todetermine the overall optimal composition or formulation for improvedtissue transport, including without limitation, transdermal transport.Specific embodiments of this invention are described in detail below.

[0017] In one embodiment, the invention concerns an apparatus formeasuring transfer of components across a tissue, comprising a supportplate, an array of samples supported by the support plate, a membrane ortissue specimen overlaying the array of samples, and a reservoir platesecured to a side of the membrane or tissue specimen opposite the arrayof samples. In one aspect of the invention, each sample in the arraycontains a unique composition or formulation of components, whereindifferent active components or different physical states of an activecomponent are present in one or more of the samples in the sample array.

[0018] In another aspect of the present invention, each sample of thearray includes a component-in-common and at least one additionalcomponent, wherein each sample differs from at least one other samplewith respect to at least one of:

[0019] (i) the identity of the additional components,

[0020] (ii) the ratio of the component-in-common to the additionalcomponents, or

[0021] (iii) the physical state of the component-in-common.

[0022] A “component-in-common” is a component that is present in everysample in a sample array. In one embodiment, the component-in-common isan active component, and preferably, the active component is apharmaceutical, dietary supplement, alternative medicine or anutraceutical. The samples may be in the form of liquids, solutions,suspensions, emulsions, solids, semi-solids, gels, foams, pastes,ointments, or triturates.

[0023] In another embodiment, the invention concerns a method ofmeasuring tissue barrier transport of a sample, comprising:

[0024] (a) preparing an array of samples having an active component andat least one additional component, wherein each sample differs from atleast one other sample with respect to at least one of:

[0025] (i) the identity of the active component;

[0026] (ii) the identity of the additional components,

[0027] (iii) the ratio of the active component to the additionalcomponents, or

[0028] (iv) the physical state of the active component;

[0029] (b) overlaying the array of samples with a tissue specimen;

[0030] (c) securing a reservoir plate to a side of the tissue specimenopposite the array of samples, the plate having an array of reservoirscorresponding to the array of samples;

[0031] (d) filling the array of reservoirs with a reservoir medium; and

[0032] (e) measuring concentration of the active component in eachreservoir at one or more time points to determine transport of theactive component from each sample across the tissue specimen.

[0033] In a preferred embodiment, the active component is apharmaceutical, a dietary supplement, an alternative medicine, or anutraceutical. In another embodiment, the tissue specimen is skin.

[0034] In another embodiment, the invention concerns a method ofanalyzing or measuring flux of a sample across a tissue, comprising:

[0035] (a) preparing an array of samples having a component-in-commonand at least one additional component, wherein each sample differs fromat least one other sample with respect to at least one of:

[0036] (i) the identity of an active component;

[0037] (ii) the identity of the additional components,

[0038] (iii) the ratio of the component-in-common to the additionalcomponents, or

[0039] (iv) the physical state of the component-in-common;

[0040] (b) overlaying the array of samples with a tissue specimen;

[0041] (c) securing a reservoir plate to a side of the tissue specimenopposite the array of samples, the plate having an array of reservoirscorresponding to the array of samples;

[0042] (d) filling the array of reservoirs with a reservoir medium; and

[0043] (e) measuring concentration of the component-in-common in eachreservoir as a function of time to determine flux of thecomponent-in-common from each sample across the tissue specimen.

[0044] In an alternative embodiment, the method comprises an additionalstep of cutting the tissue specimen to avoid lateral diffusion betweenwells. The method preferably comprises analyzing the tissue specimen fordefects, or inhomogeneities, and correcting for or repairing thedefects.

[0045] According to the inventon there is provided a transdermal assayapparatus. The transdermal assay apparatus includes at least first andsecond members. The first member has one or more sample surfaces, eachof which is configured to receive a sample thereon. The second memberdefines one or more reservoirs, each of which has an opening on asurface of the second member. Each sample surface is substantially thesame size as each opening. The transdermal assay apparatus also includesa magnetic clamp configured to clamp a tissue specimen between thesample surface and the opening. The magnetic clamp also seals thevarious members of the apparatus together. The magnetic clamp preferablyincludes a magnet having a strength that is selected based on theclamping force required between the first member and the second member.

[0046] The invention also provides a method for using a transdermalassay apparatus. A sample is placed on a sample surface of a firstmember. A reservoir defined by a second member is filled with a fluidmedium, where the reservoir has an opening on a surface of the secondmember. A tissue specimen is then placed between the sample and theopening. The tissue specimen is subsequently clamped between the sampleand the fluid medium at the opening, with a magnetic clamping force.Periodically, a specimen of the fluid medium is withdrawn from thereservoir to measure the concenration of the sample in the fluid medium.This concentration is indicative of a transfer of the sample across thetissue specimen.

[0047] The above described apparatus and method are preferably used forhigh-throughput screening of active component or drug flux through thestratum corneum recognizing that such flux is determined, at least inpart, by the permeability of the drug within the tissue in the presenceof an enhancer. The permeability is generally governed by at least twofactors: the solubility of the active component or drug within thestratum corneum and the diffusivity of the active component or drugwithin the stratum corneum. These two factors, solubility anddiffusivity, can be measured independently as a method of indirectlyassessing the flux through the stratum corneum. Thus, an array of wellscontaining samples of different compositions of active component andinactive compounds, including without limitation, compositionscomprising active component/carrier or excipient/, activecomponent/carrier or excipient/enhancer/, activecomponent/adhesive/enhancer/additive, are constructed. Known amounts ofstratum corneum are added to each well and the rate at which the activecomponent or drug is taken up into the tissue sample is measured byextracting the tissue from similarly prepared wells at different times.Measuring the concentration after times sufficiently long so that theamount dissolved is not changing with time can assess the equilibriumconcentration of active component or drug within the tissue. The productof the rate and solubility is proportional to the permeability of theactive component or drug.

[0048] The high-throughput combinatorial screening systems and methodsof the present invention identify optimal compositions or formulationsto achieve a desired result for such compositions or formulations,including without limitation, construction of a transdermal deliverydevice. In particular, the systems and methods of the present inventionmay be used to identify 1) optimal compositions or formulationscomprising one or more active components and one or more inactivecomponents for achieving desired characteristics for such compositionsor formulations, 2) optimal adhesive/enhancer/additive compositions forcompatibility with a drug, 3) optimal drug/adhesive/enhancer/additivecompositions for maximum drug flux through stratum corneum, and 4)optimal drug/adhesive/enhancer/additive composition to minimizecytotoxicity

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The features and advantages of the present invention will bebetter understood by reference to the following detailed description,which should be read in conjunction with the accompanying drawings inwhich:

[0050]FIG. 1 is a schematic diagram of a high-throughput apparatus formeasuring tissue barrier transport, such as transdermal transport,according to the present invention;

[0051]FIGS. 2A-2D are schematic diagrams of an alternative embodiment ofa high-throughput apparatus for measuring tissue barrier transport usingsolid source samples according to the present invention;

[0052]FIG. 3 is a schematic diagram of an alternative embodiment of ahigh-throughput apparatus for measuring tissue barrier transportaccording to the present invention;

[0053]FIGS. 4A-4C are schematic diagrams of an alternative embodiment ofa diffusion cell that alone, or as part of a high throughput apparatus,is used for measuring tissue barrier transport according to the presentinvention.

[0054]FIGS. 5A and 5B are schematic diagrams of an apparatus for fillinga sample well in a sample array, such as the sample array in thehigh-throughput apparatus shown in FIG. 1;

[0055]FIG. 6 is a schematic diagram of an alternative embodiment of ahigh-throughput apparatus for measuring or analyzing tissue barriertransport using solid source samples according to the present invention;

[0056]FIG. 7 is a schematic diagram of an alternative embodiment of ahigh-throughput apparatus for measuring or analyzing tissue barriertransport using solid source samples according to the present invention;

[0057]FIG. 8 is a schematic diagram of an alternative embodiment of ahigh-throughput apparatus for measuring or analyzing tissue barriertransport using solid source samples according to the present invention;

[0058]FIG. 9 is an exploded view of a another transdermal assayapparatus according to another embodiment of the invention; and

[0059]FIG. 10 is an exploded view of a yet another transdermal assayapparatus according to yet another embodiment of the invention.

5. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention relates to high throughput combinatorialsystems and methods that improve tissue barrier transfer of activecompounds, such as pharmaceuticals or drugs, other compounds, orcompound combinations. In one embodiment, the system and methods of thepresent invention may be used to identify the optimal components (e.g.,solvents, carriers, transport enhancers, adhesives, additives, and otherexcipients) for pharmaceutical compositions or formulations that aredelivered to a patient via tissue transport, including withoutlimitation, pharmaceutical compositions or formulations administered ordelivered transdermally (e.g., in the form of a transdermal deliverydevice), topically (e.g., in the form of ointments, lotions, gels, andsolutions), and ocularly (e.g., in the form of a solution). As usedherein, “high throughput” refers to the number of samples generated orscreened as described herein, typically at least 10, more typically atleast 50 to 100, and preferably more than 1000 samples.

[0061] The high throughput methods of the present invention can beperformed using various forms of samples. Typically, the methods areperformed either with liquid samples or with solid or semi-solidsamples.

[0062] As used herein, “liquid source” means that the sample containingthe component or components being measured or analyzed is in the form ofa liquid, which includes, without limitation, liquids, solutions,emulsions, suspensions, and any of the foregoing having solidparticulates dispersed therein.

[0063] As used herein, “solid source” means that the sample containingthe component or components being measured or analyzed is in the form ofa solid or semi-solid, which includes, without limitation, triturates,gels, films, foams, pastes, ointments, adhesives, high viscoelasticliquids, high viscoelastic liquids having solid particulates dispersedtherein, and transdermal patches.

[0064] As used herein, “reservoir medium” or “fluid medium” refers to aliquid, solution, gel, or sponge that is chemically compatible with thecomponents in a sample and the tissue being used in an apparatus ormethod of the present invention. In one embodiment of the presentinvention, the reservoir medium comprises part of the specimen taken tomeasure or analyze the transfer, flux, or diffusion of a componentacross a tissue barrier. Preferably, the reservoir medium is a liquid orsolution.

[0065] 5.1 Overview of an Apparatus for Measuring Tissue BarrierTransfer

[0066]FIG. 1 shows a schematic diagram of a preferred embodiment of ahigh-throughput apparatus 100 for measuring tissue barrier transport ina sample array 112 according to the present invention. Apparatus 100includes a substrate plate 114 supporting sample array 112, a tissuespecimen 120 and a reservoir plate 130. In this embodiment, each samplein sample array 112 is placed in a sample well 116. Attached to thebottom of substrate plate 114 is a base 118 that forms the bottom ofeach sample well 116. Base 118 is optionally a membrane made of anysuitable material (e.g. a rubber membrane) in any fashion that permitsair to bleed out of sample well 116 when filling with a sample.Alternatively, base 118 is a rigid, removable substrate plate such asplate 214 (described infra with respect to FIGS. 2A-2D) capable ofsupporting an array of solid source samples.

[0067] Substrate plate 114 may be any rigid grid or plate capable ofsupporting a number of samples. For example, substrate plate 114 may bea 24, 36, 48, 72, 96 or 384 well plate. Preferably, apparatus 100comprises one or more sample arrays 112, wherein the number of samplewells 116 in apparatus 100 is at least 100, preferably at least 1000,and more preferably at least 10,000. Preferably, the size of sample well116 is about 1 mm to about 50 mm, more preferably about 2 mm to about 10mm, and most preferably about 3 mm to about 7 mm. For example, a 3 mmwell format provides an array of approximately 30,000 samples for 0.25m² of skin.

[0068] As used herein, the terms “array” or “sample array” (e.g. array112) mean a plurality of samples associated under a common experiment,wherein each of the samples comprises at least two components, and atleast one of the components being an active component. In one embodimentof the present invention, one of the sample components is a“component-in-common”, which as used herein, means a component that ispresent in every sample of the array, with the exception of negativecontrols.

[0069] Sample array 112 is designed to provide a number of differentsamples of different compositions, the analysis of which allowsdetermination of optimal compositions or formulations for improvingtransfer of a component across tissue 120. Each sample in sample array112 preferably, though not necessarily, differs from any other sample inthe array with respect to at least one of:

[0070] (i) the identity of the active component;

[0071] (ii) the identity of the additional component;

[0072] (iii) the ratio of the active component, or thecomponent-in-common, to the additional component; or

[0073] (iv) the physical state of the active component, or thecomponent-in-common.

[0074] An array can comprise 24, 36, 48, 96, or more samples, preferablyat least 1000 samples, more preferably, at least 10,000 samples. Anarray is typically comprised of one or more sub-arrays. For example, asub-array can be a plate having 96 sample wells.

[0075] Overlaying substrate plate 114 and sample array 112 is tissuespecimen 120. Tissue 120 is preferably a sheet of tissue, such as skin,lung, tracheal, nasal, placental, vaginal, rectal, colon, gut, stomach,bladder, or corneal tissue. More preferably, tissue 120 is skin tissueor stratum corneum. If human cadaver skin is to be used for tissue 120,one known method of preparing the tissue specimen entails heat strippingby keeping it in water at 60° C. for two minutes followed by the removalof the epidermis, and storage at 4° C. in a humidified chamber. A pieceof epidermis is taken out from the chamber prior to the experiments andplaced over substrate plate 114. Tissue 120 is optionally be supportedby Nylon mesh (Terko Inc.) to avoid any damage and to mimic the factthat the skin in vivo is supported by mechanically strong dermis.Alternatively, other types of tissues may be used, including livingtissue explants, animal tissue (e.g. rodent, bovine or swine) orengineered tissue-equivalents. Examples of a suitable engineered tissuesinclude DERMAGRAFT (Advanced Tissue Sciences, Inc.) and those taught inU.S. Pat. No. 5,266,480, which is incorporated herein by reference.

[0076] In an alternative embodiment of the present invention, tissuespecimen 120 is divided into a number of segments by cuts 122 betweensample wells 116 to prevent lateral diffusion through tissue specimen120 between adjacent samples. Cuts 122 may be made in any number ofways, including mechanical scribing or cutting, laser cutting, orcrimping (e.g., between plates 114 and 130 or by using a “waffle iron”type embossing tool). Preferably, laser scribing is used as it avoidsmechanical pressure from a cutting tool which can cause distortion anddamage to tissue specimen 120. Laser cuts 122 are performed with verysmall kerfs which permit a relatively high density of samples and a moreefficient tissue specimen utilization. Laser tools are available thatproduce a minimal heat affected zone, thereby reducing damage to tissuespecimen 120.

[0077] Reservoir plate 130 (e.g., an open-bottomed titer plate) isplaced on top of tissue 120, on a side of tissue opposite substrateplate 114. Reservoir plate includes a number of hollow reservoirs 132.When plate 130 is secured in place, each reservoir 132 aligns over asample well 116 such that tissue separates each well 116 from reservoir132. Reservoir plate 130 secures to substrate plate 114 using clamps,screws, fasteners, or any other suitable attachment means. Plates 130and 114 preferably secure together with sufficient pressure so as tocreate a liquid tight seal around reservoirs 132. Each reservoir isfilled with a reservoir, such as a saline solution, to receive samplecomponents or compounds that diffuse across tissue 120 to reservoir 132.In one embodiment, the reservoir medium is approximately 2% BSA solutionin PBS.

[0078] Transfer or flux of components from sample wells 116 acrosstissue 120 (i.e., tissue barrier transfer or diffusion) may be analyzedby measuring component concentration in specimens taken from reservoirs132. Comparison of measurements taken from different samples/reservoirsaids in determining optimal sample compositions for improving tissuetransfer or diffusion of a desired component (e.g,. a pharmaceutical).

[0079] Preferably the samples are prepared, added to sample wells andmixed automatically. Similarly, specimen from reservoirs 132 containingtransferred or diffused components, and the concentrations thereof, canbe measured and processed automatically. “Automated” or “automatically”refers to the use of computer software and robotics to add, mix andanalyze the samples, components, and specimens or diffusion products.

[0080] Samples are added to the sample wells in sample arrays of thepresent invention, such as sample array 112 in FIG. 1, using variousdeposition or material transfer techniques known to the skilled artisan,including, without limitation, hand placement, pipetting, and othermanual or automated solid or liquid distribution systems.

[0081] After adding and mixing the components to the sample wells, thesamples may be processed by well known techniques, such as heating,filtration, and lyophilization. One of skill in the art will know how toprocess the sample according to the properties being tested. The samplescan be processed individually or as a group, preferably, as a group.Additional details regarding suitable automated dispensing and samplingequipment and methods of formulating solutions or compositions aredisclosed in copending U.S. patent application Ser. No. 09/540,462 whichis herein incorporated by reference in its entirety.

[0082] Briefly, a number of companies have developed microarray systemsthat can be adapted for use in the system described herein, although allare currently used for the sole purpose of screening to identifycompounds having a particular defined activity, as opposed to screeningof components or compounds having a known identity in order to identifyoptimal component combinations to achieve a desired result. Such systemsmay require modification, which is well within ordinary skill in theart. Examples of companies having microarray systems include Gene Logicof Gaithersburg, Md. (see U.S. Pat. No. 5,843,767 to Beattie), LuminexCorp., Austin, Tex., Beckman Instruments, Fullerton, Calif., MicroFabTechnologies, Plano, Tex., Nanogen, San Diego, Calif., and Hyseq,Sunnyvale, Calif. These devices test samples based on a variety ofdifferent systems. All include thousands of microscopic channels thatdirect components into test wells, where reactions can occur. Thesesystems are connected to computers for analysis of the data usingappropriate software and data sets. The Beckman Instruments system candeliver nanoliter samples of 96 or 384-arrays, and is particularly wellsuited for hybridization analysis of nucleotide molecule sequences. TheMicroFab Technologies system delivers sample using inkjet printers toaliquot discrete samples into wells.

[0083] These and other systems can be adapted as required for useherein. For example, the combinations of active component and variousadditional or inactive components at various concentrations andcombinations can be generated using standard formulating software (e.g.,Matlab software, commercially available from Mathworks, Natick, Mass.).The combinations thus generated can be downloaded into a spread sheet,such as Microsoft EXCEL. From the spread sheet, a work list can begenerated for instructing the automated distribution mechanism toprepare an array of samples according to the various combinationsgenerated by the formulating software. The work list can be generatedusing standard programming methods according to the automateddistribution mechanism that is being used. The use of so-called worklists simply allows a file to be used as the process command rather thandiscrete programmed steps. The work list combines the formulation outputof the formulating program with the appropriate commands in a fileformat directly readable by the automatic distribution mechanism.

[0084] The automated distribution mechanism delivers at least one activecomponent, such as a pharmaceutical, as well as various inactive oradditional components, such as solvents, carriers, excipients, andadditives, to each sample well. Preferably, the automated distributionmechanism can deliver multiple amounts of each component. In oneembodiment, the automated distribution mechanism utilizes one or moremicro-solenoid valves.

[0085] Automated liquid and solid distribution systems are well knownand commercially available, such as the Tecan Genesis, from Tecan-US,RTP, North Carolina. The robotic arm can collect and dispense activecomponents and inactive components, such as solutions, solvents,carriers, excipients, additives, and the like, from a stock plate to asample well or site. The process is repeated until an array iscompleted. The samples are then mixed. For example, the robotic armmoves up and down in each well plate for a set number of times to ensureproper mixing.

[0086] In use, apparatus 100 of FIG. 1 is described above as havingreservoir medium above tissue 120 in reservoirs 132 and samples belowtissue 120 in sample wells 116 of array 112. In an alternativeembodiment, the positions are reversed, such that reservoirs 132 ofsample array 112 are below tissue specimen 120 and sample wells 116 areabove tissue specimen 120, and a top plate or top membrane is situatedover reservoirs 132 and reservoir plate 130.

[0087] Additional embodiments of the systems and methods of the presentinvention are described infra, particularly with respect to FIGS. 2-8.

[0088] 5.2 Composition of Samples

[0089] Before discussing additional details of the systems and methodsfor assessing tissue barrier transfer according to the presentinvention, applicants present a discussion of the composition of samplessuitable for use in the present invention.

[0090] 5.2.1 General Composition Terminology

[0091] As used herein, the term “component” means any substance orcompound. A component can be active or inactive. As used herein, theterm “active component” means a substance or compound that imparts aprimary utility to a composition or formulation when the composition orformulation is used for its intended purpose. Examples of activecomponents include pharmaceuticals, dietary supplements, alternativemedicines, and nutraceuticals. Active components can optionally besensory compounds, agrochemicals, the active component of a consumerproduct formulation, or the active component of an industrial productformulation. As used herein, an “inactive component” means a componentthat is useful or potentially useful to serve in a composition orformulation for administration of an active component, but does notsignificantly share in the active properties of the active component orgive rise to the primary utility for the composition or formulation.Examples of suitable inactive components include, but are not limitedto, enhancers, excipients, carriers, solvents, diluents, stabilizers,additives, adhesives, and combinations thereof.

[0092] Preferably, the samples of an array comprise an active componentand inactive components. In one embodiment, the active components in thesamples of an array can be the same or different, while in anotherembodiment, the samples in an array comprise an active component as acomponent-in-common and inactive components. A number of permutationsare available to the skilled artisan, for example, when the activecomponent is a pharmaceutical, dietary supplement, alternative medicine,or nutraceutical, the preferred inactive components are selected fromthe group consisting of excipients, carriers, solvents, diluents,stabilizers, enhancers, additives, adhesives, and combinations thereof.

[0093] As used herein, the term “sample” means a mixture of an activecomponent and one or more additional components or inactive components.Preferably a sample comprises 2 or more additional components, morepreferably, 3 or more additional components. In general, a sample willcomprise one active component but can comprise multiple activecomponents. In addition, samples in a sample array may have one or morecomponents-in-common. A sample can be present in any container or holderor in or on any material or surface, the only requirement is that thesamples be located at separate sites. Preferably, samples are containedin sample wells, for example, a 24, 36, 48, or 96 well plates (or filterplates) of volume 250 μl available from Millipore, Bedford, Mass. Thesample can comprise less than about 100 milligrams of the activecomponent, preferably, less than about 1 milligram, more preferably,less than about 100 micrograms, and even more preferably, less than 100nanograms. Preferably, the sample has a total volume of about 1-200 μl,more preferably about 5-150 μl, and most preferably about 10-100 μl.Samples can be liquid source or solid source samples, which includesamples in the form of solids, semi-solids, films, liquids, solutions,gels, foams, pastes, ointments, triturates, suspensions, or emulsions.

[0094] According to the invention described herein, the “physical state”of a component is initially defined by whether the component is a liquidor a solid. If a component is a solid, the physical state is furtherdefined by the particle size and whether the component is crystalline oramorphous. If the component is crystalline, the physical state isfurther divided into: (1) whether the crystal matrix includes aco-adduct or whether the crystal matrix originally included a co-adduct,but the co-adduct was removed leaving behind a vacancy; (2) crystalhabit; (3) morphology, i.e., crystal habit and size distribution; and(4) internal structure (polymorphism). In a co-adduct, the crystalmatrix can include either a stoichiometric or non-stoichiometric amountof the adduct, for example, a crystallization solvent or water, i.e., asolvate or a hydrate. Non-stoichiometric solvates and hydrates includeinclusions or clathrates, that is, where a solvent or water is trappedat random intervals within the crystal matrix, for example, in channels.A stoichiometric solvate or hydrate is where a crystal matrix includes asolvent or water at specific sites in a specific ratio. That is, thesolvent or water molecule is part of the crystal matrix in a definedarrangement. Additionally, the physical state of a crystal matrix canchange by removing a co-adduct, originally present in the crystalmatrix. For example, if a solvent or water is removed from a solvate ora hydrate, a hole will be formed within the crystal matrix, therebyforming a new physical state. The crystal habit is the description ofthe outer appearance of an individual crystal, for example, a crystalmay have a cubic, tetragonal, orthorhombic, monoclinic, triclinic,rhomboidal, or hexagonal shape. The processing characteristics areaffected by crystal habit. The internal structure of a crystal refers tothe crystalline form or polymorphism. A given compound may exist asdifferent polymorphs, that is, distinct crystalline species. In general,different polymorphs of a given compound are as different in structureand properties as the crystals of two different compounds. Solubility,melting point, density, hardness, crystal shape, optical and electricalproperties, vapor pressure, and stability, etc. all vary with thepolymorphic form.

[0095] 5.2.2 Active Component and Component-In-Common

[0096] As mentioned above, the component-in-common can be either anactive component, such as a pharmaceutical, dietary supplement,alternative medicine, or nutraceutical, or an inactive component. In apreferred embodiment of the present invention, the component-in-commonis an active component, and more preferably a pharmaceutical. As usedherein, the term “pharmaceutical” means any substance or compound thathas a therapeutic, disease preventive, diagnostic, or prophylacticeffect when administered to an animal or a human. The termpharmaceutical includes prescription drugs and over the counter drugs.Pharmaceuticals suitable for use in the invention include all thoseknown or to be developed.

[0097] Examples of suitable pharmaceuticals include, but are not limitedto, cardiovascular pharmaceuticals, such as amlodipine besylate,losartan potassium, irbesartan, diltiazem hydrochloride, clopidogrelbisulfate, digoxin, abciximab, furosemide, amiodarone hydrochloride,beraprost, tocopheryl nicotinate; anti-infective components, such asamoxicillin, clavulanate potassium, azithromycin, itraconazole,acyclovir, fluconazole, terbinafine hydrochloride, erythromycinethylsuccinate, and acetyl sulfisoxazole; psychotherapeutic components,such as sertraline hydrochloride, venlafaxine, bupropion hydrochloride,olanzapine, buspirone hydrochloride, alprazolam, methylphenidatehydrochloride, fluvoxamine maleate, and ergoloid mesylates;gastrointestinal products, such as lansoprazole, ranitidinehydrochloride, famotidine, ondansetron hydrochloride, granisetronhydrochloride, sulfasalazine, and infliximab; respiratory therapies,such as loratadine, fexofenadine hydrochloride, cetirizinehydrochloride, fluticasone propionate, salmeterol xinafoate, andbudesonide; cholesterol reducers, such as atorvastatin calcium,lovastatin, bezafibrate, ciprofibrate, and gemfibrozil; cancer andcancer-related therapies, such as paclitaxel, carboplatin, tamoxifencitrate, docetaxel, epirubicin hydrochloride, leuprolide acetate,bicalutamide, goserelin acetate implant, irinotecan hydrochloride,gemcitabine hydrochloride, and sargramostim; blood modifiers, such asepoetin alfa, enoxaparin sodium, and antihemophilic factor;antiarthritic components, such as celecoxib, nabumetone, misoprostol,and rofecoxib; AIDS and AIDS-related pharmaceuticals, such aslamivudine, indinavir sulfate, stavudine, and lamivudine; diabetes anddiabetes-related therapies, such as metformin hydrochloride,troglitazone, and acarbose; biologicals, such as hepatitis B vaccine,and hepatitis A vaccine; hormones, such as estradiol, mycophenolatemofetil, and methylprednisolone; analgesics, such as tramadolhydrochloride, fentanyl, metamizole, ketoprofen, morphine sulfate,lysine acetylsalicylate, ketorolac tromethamine, morphine, loxoprofensodium, and ibuprofen; dermatological products, such as isotretinoin andclindamycin phosphate; anesthetics, such as propofol, midazolamhydrochloride, and lidocaine hydrochloride; migraine therapies, such assumatriptan succinate, zolmitriptan, and rizatriptan benzoate; sedativesand hypnotics, such as zolpidem, zolpidem tartrate, triazolam, andhycosine butylbromide; imaging components, such as iohexol, technetium,TC99M, sestamibi, iomeprol, gadodiamide, ioversol, and iopromide; anddiagnostic and contrast components, such as alsactide, americium,betazole, histamine, mannitol, metyrapone, petagastrin, phentolamine,radioactive B₁₂, gadodiamide, gadopentetic acid, gadoteridol, andperflubron. Other pharmaceuticals for use in the invention include thoselisted in Table 1 below, which suffer from problems that could bemitigated by developing new compositions or formulations using thesystems, arrays and methods of the present invention. TABLE 1 ExemplaryPharmaceuticals Brand Name Chemical Properties SANDIMMINE cyclosporinPoor absorption due to its low water solubility. TAXOL paclitaxel Poorabsorption due to its low water solubility. VIAGRA sildenafil citratePoor absorption due to its low water solubility. NORVIR ritonavir Canundergo a polymorphic shift during shipping and storage. FULVICINgriseofulvin Poor absorption due to its low water solubility. FORTOVASEsaquinavir Poor absorption due to its low water solubility.

[0098] Still other examples of suitable pharmaceuticals are listed in2000 Med Ad News 19:56-60 and The Physicians Desk Reference, 53rdedition, 792-796, Medical Economics Company (1999), both of which areincorporated herein by reference.

[0099] Examples of suitable veterinary pharmaceuticals include, but arenot limited to, vaccines, antibiotics, growth enhancing components, anddewormers. Other examples of suitable veterinary pharmaceuticals arelisted in The Merck Veterinary Manual, 8th ed., Merck and Co., Inc.,Rahway, N.J., 1998; (1997); The Encyclopedia of Chemical Technology, 24Kirk-Othomer (4^(th) ed. at 826); and Veterinary Drugs in ECT 2nd ed.,Vol 21, by A. L. Shore and R. J. Magee, American Cyanamid Co.

[0100] Other active components suitable for tissue (or trans-membrane)transfer analysis using the systems and methods of the present inventioninclude dietary supplements, alternative medicines, or nutraceuticals.

[0101] As used herein, the term “dietary supplement” means a non-caloricor insignificant-caloric substance administered to an animal or a humanto provide a nutritional benefit or a non-caloric orinsignificant-caloric substance administered in a food to impart thefood with an aesthetic, textural, stabilizing, or nutritional benefit.Dietary supplements include, but are not limited to, fat binders, suchas caducean; fish oils; plant extracts, such as garlic and pepperextracts; vitamins and minerals; food additives, such as preservatives,acidulents, anticaking components, antifoaming components, antioxidants,bulking components, coloring components, curing components, dietaryfibers, emulsifiers, enzymes, firming components, humectants, leaveningcomponents, lubricants, non-nutritive sweeteners, food-grade solvents,thickeners; fat substitutes, and flavor enhancers; and dietary aids,such as appetite suppressants. Examples of suitable dietary supplementsare listed in (1994) The Encyclopedia of Chemical Technology, 11Kirk-Othomer (4^(th) ed. at 805-833). Examples of suitable vitamins arelisted in (1998) The Encyclopedia of Chemical Technology, 25Kirk-Othomer (4^(th) ed. at 1) and Goodman & Gilman 's: ThePharmacological Basis of Therapeutics, 9th Edition, eds. Joel G. Harmanand Lee E. Limbird, McGraw-Hill, 1996 p. 1547, both of which areincorporated by reference herein. Examples of suitable minerals arelisted in The Encyclopedia of Chemical Technology, 16 Kirk-Othomer(4^(th) ed. at 746) and “Mineral Nutrients” in ECT 3rd ed., Vol 15, pp.570-603, by C. L. Rollinson and M. G. Enig, University of Maryland, bothof which are incorporated herein by reference

[0102] As used herein, the term “alternative medicine” means asubstance, preferably a natural substance, such as a herb or an herbextract or concentrate, administered to a subject or a patient for thetreatment of disease or for general health or well being, wherein thesubstance does not require approval by the FDA. Examples of suitablealternative medicines include, but are not limited to, ginkgo biloba,ginseng root, valerian root, oak bark, kava kava, echinacea,harpagophyti radix, others are listed in The Complete German CommissionE Monographs: Therapeutic Guide to Herbal Medicine, Mark Blumenthal etal. eds., Integrative Medicine Communications 1998, incorporated byreference herein.

[0103] As used herein the term “nutraceutical” means a food or foodproduct having both caloric value and pharmaceutical or therapeuticproperties. Example of nutraceuticals include garlic, pepper, brans andfibers, and health drinks Examples of suitable Nutraceuticals are listedin M. C. Linder, ed. Nutritional Biochemistry and Metabolism withClinical Applications, Elsevier, New York, 1985; Pszczola et al., 1998Food technology 52:30-37 and Shukla et al., 1992 Cereal Foods World37:665-666.

[0104] Preferably, when the active component is a pharmaceutical, adietary supplement, an alternative medicine, or a nutraceutical, atleast one additional component(s) is an excipient. As used herein, theterm “excipient” means the inactive substances used to formulatepharmaceuticals as a result of processing or manufacture or used bythose of skill in the art to formulate pharmaceuticals, dietarysupplements, alternative medicines, and nutraceuticals foradministration to animals or humans. Preferably, excipients are approvedfor or considered to be safe for human and animal administration.Examples of suitable excipients include, but are not limited to,acidulents, such as lactic acid, hydrochloric acid, and tartaric acid;solubilizing components, such as non-ionic, cationic, and anionicsurfactants; absorbents, such as bentonite, cellulose, and kaolin;alkalizing components, such as diethanolamine, potassium citrate, andsodium bicarbonate; anticaking components, such as calcium phosphatetribasic, magnesium trisilicate, and talc; antimicrobial components,such as benzoic acid, sorbic acid, benzyl alcohol, benzethoniumchloride, bronopol, alkyl parabens, cetrimide, phenol, phenylmercuricacetate, thimerosol, and phenoxyethanol; antioxidants, such as ascorbicacid, alpha tocopherol, propyl gallate, and sodium metabisulfite;binders, such as acacia, alginic acid, carboxymethyl cellulose,hydroxyethyl cellulose; dextrin, gelatin, guar gum, magnesium aluminumsilicate, maltodextrin, povidone, starch, vegetable oil, and zein;buffering components, such as sodium phosphate, malic acid, andpotassium citrate; chelating components, such as EDTA, malic acid, andmaltol; coating components, such as adjunct sugar, cetyl alcohol,polyvinyl alcohol, carnauba wax, lactose maltitol, titanium dioxide;controlled release vehicles, such as microcrystalline wax, white wax,and yellow wax; desiccants, such as calcium sulfate; detergents, such assodium lauryl sulfate; diluents, such as calcium phosphate, sorbitol,starch, talc, lactitol, polymethacrylates, sodium chloride, and glycerylpalmitostearate; disintegrants, such as collodial silicon dioxide,croscarmellose sodium, magnesium aluminum silicate, potassiumpolacrilin, and sodium starch glycolate; dispersing components, such aspoloxamer 386, and polyoxyethylene fatty esters (polysorbates);emollients, such as cetearyl alcohol, lanolin, mineral oil, petrolatum,cholesterol, isopropyl myristate, and lecithin; emulsifying components,such as anionic emulsifying wax, monoethanolamine, and medium chaintriglycerides; flavoring components, such as ethyl maltol, ethylvanillin, fumaric acid, malic acid, maltol, and menthol; humectants,such as glycerin, propylene glycol, sorbitol, and triacetin; lubricants,such as calcium stearate, canola oil, glyceryl palmitosterate, magnesiumoxide, poloxymer, sodium benzoate, stearic acid, and zinc stearate;solvents, such as alcohols, benzyl phenylformate, vegetable oils,diethyl phthalate, ethyl oleate, glycerol, glycofurol, for indigocarmine, polyethylene glycol, for sunset yellow, for tartazine,triacetin; stabilizing components, such as cyclodextrins, albumin,xanthan gum; and tonicity components, such as glycerol, dextrose,potassium chloride, and sodium chloride; and mixture thereof. Excipientsinclude those that alter the rate of absorption, bioavailability, orother pharmacokinetic properties of pharmaceuticals, dietarysupplements, alternative medicines, or nutraceuticals. Other examples ofsuitable excipients, such as binders and fillers are listed in Remington's Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, MackPublishing Co. Easton, Pa., 1995 and Handbook of PharmaceuticalExcipients, 3rd Edition, ed. Arthur H. Kibbe, American PharmaceuticalAssociation, Washington D.C. 2000, both of which are incorporated hereinby reference.

[0105] Excipients that are typically used in the formation oftransdermal delivery devices, and therefore particularly useful forformulation of the samples of the present invention, are penetrationenhancers, adhesives and solvents. Each of these is discussed in moredetail below.

[0106] 5.2.3 Penetration Enhancers

[0107] Various types of penetration enhancers may be used to enhancetransdermal transport of drugs. Penetration enhancers can be dividedinto chemical enhancers and mechanical enhancers, each of which isdescribed in more detail below.

[0108] 5.2.3.1 Chemical Enhancers

[0109] Chemical enhancers enhance molecular transport rates acrosstissues or membranes by a variety of mechanisms. In the presentinvention, chemical enhancers are preferably used to decrease thebarrier properties of the stratum corneum. Drug interactions includemodifying the drug into a more permeable state (a prodrug), which wouldthen be metabolized inside the body back to its original form(6-fluorouracil, hydrocortisone) (Hadgraft, 1985); or increasing drugsolubilities (ethanol, propylene glycol). Despite a great deal ofresearch (well over 200 compounds have been studied) (Chattaraj andWalker, 1995), there are still no universally applicable mechanistictheories for the chemical enhancement of molecular transport. Most ofthe published work in chemical enhancers has been done largely based onexperience and on a trial-and-error basis (Johnson, 1996).

[0110] Many different classes of chemical enhancers have beenidentified, including cationic, anionic, and nonionic surfactants(sodium dodecyl sulfate, polyoxamers); fatty acids and alcohols(ethanol, oleic acid, lauric acid, liposomes); anticholinergic agents(benzilonium bromide, oxyphenonium bromide); alkanones (n-heptane);amides (urea, N,N-diethyl-m-toluamide); fatty acid esters (n-butyrate);organic acids (citric acid); polyols (ethylene glycol, glycerol);sulfoxides (dimethylsulfoxide); and terpenes (cyclohexene) (Hadgraft andGuy, 1989; Walters, 1989; Williams and Barry, 1992; Chattaraj andWalker, 1995). Most of these enhancers interact either with the skin orwith the drug. Those enhancers interacting with the skin are hereintermed “lipid permeation enhancers”, and include interactions with theskin include enhancer partitioning into the stratum corneum, causingdisruption of the lipid bilayers (azone, ethanol, lauric acid), bindingand disruption of the proteins within the stratum corneum (sodiumdodecyl sulfate, dimethyl sulfoxide), or hydration of the lipid bilayers(urea, benzilonium bromide). Other chemical enhancers work to increasethe transdermal delivery of a drug by increasing the drug solubility inits vehicle (hereinafter termed “solubility enhancers”). Lipidpermeation enhancers, solubility enhancers, and combinations ofenhancers (also termed “binary systems”) are discussed in more detailbelow.

5.2.3.1.1 Lipid Permeation Enhancers

[0111] Chemicals which enhance permeability through lipids are known andcommercially available. For example, ethanol increases the solubility ofdrugs up to 10,000-fold and yield a 140-fold flux increase of estradiol,while unsaturated fatty acids increase the fluidity of lipid bilayers(Bronaugh and Maibach, editors (Marcel Dekker 1989) pp. 1-12. Examplesof fatty acids which disrupt lipid bilayer include linoleic acid, capricacid, lauric acid, and neodecanoic acid, which can be in a solvent suchas ethanol or propylene glycol. Evaluation of published permeation datautilizing lipid bilayer disrupting agents agrees very well with theobservation of a size dependence of permeation enhancement forlipophilic compounds. The permeation enhancement of three bilayerdisrupting compounds, capric acid, lauric acid, and neodecanoic acid, inpropylene glycol has been reported by Aungst, et al. Pharm. Res.7,712-718 (1990). They examined the permeability of four lipophiliccompounds, benzoic acid (122 Da), testosterone (288 Da), naloxone (328Da), and indomethacin (359 Da) through human skin. The permeabilityenhancement of each enhancer for each drug was calculated according toE_(c pg)=P_(e pg)/P_(pg), where P_(e pg) is the drug permeability fromthe enhancer/propylene glycol formulation and P_(pg) is the permeabilityfrom propylene glycol alone.

[0112] The primary mechanism by which unsaturated fatty acids, such aslinoleic acid, are thought to enhance skin permeability is bydisordering the intercellular lipid domain. For example, detailedstructural studies of unsaturated fatty acids, such as oleic acid, havebeen performed utilizing differential scanning calorimetry (Barry J.Controlled Release 6,85-97 (1987)) and infrared spectroscopy(Ongpipattanankul, et al., Pharm. Res. 8, 350-354 (1991); Mark, et al.,J. Control. Rd. 12, 67-75 (1990)). Oleic acid was found to disorder thehighly ordered SC lipid bilayers, and to possibly form a separate,oil-like phase in the intercellular domain. SC Lipid bilayers disorderedby unsaturated fatty acids or other bilayer disrupters may be similar innature to fluid phase lipid bilayers.

[0113] A separated oil phase should have properties similar to a bulkoil phase. Much is known about transport a fluid bilayers and bulk oilphases. Specifically, diffusion coefficients in fluid phase, forexample, dimyristoylphosphatidylcholine (DMPC) bilayers Clegg and Vaz In“Progress in Protein-Lipid Interactions” Watts, ed. (Elsevier, N.Y.1985) 173-229; Tocanne, et al., FEB 257, 10-16 (1989) and in bulk oilphase Perry, et al., “Perry's Chemical Engineering Handbook”(McGraw-Hill, NY 1984) are greater than those in the SC, and moreimportantly, they exhibit size dependencies which are considerablyweaker than that of SC transport Kasting, et al., In: “Prodrugs: Topicaland Ocular Delivery” Sloan. ed. (Marcel Dekker, NY 1992) 117-161; Portsand Guy, Pharm. Res. 9, 663-339 (1992); Willschut, et al. Chemosphere30, 1275-1296 (1995). As a result, the diffusion coefficient of a givensolute will be greater in a fluid bilayer, such as DMPC, or a bulk oilphase than in the SC. Due to the strong size dependence of SC transport,diffusion in SC lipids is considerably slower for larger compounds,while transport in fluid DMPC bilayers and bulk oil phases is onlymoderately lower for larger compounds. The difference between thediffusion coefficient in the SC and those in fluid DMPC bilayers or bulkoil phases will be greater for larger solutes, and less for smallercompounds. Therefore, the enhancement ability of a bilayer disorderingcompound which can transform the SC lipids bilayers into a fluid bilayerphase or add a separate bulk oil phase should exhibit a size dependence,with smaller permeability enhancements for small compounds and largerenhancement for larger compounds.

[0114] A comprehensive list of lipid bilayer disrupting agents isdescribed in European Patent Application 43,738 (1982), which isincorporated herein by reference. Exemplary compounds are represented bythe formula:

[0115] R—X, wherein R is a straight-chain alkyl of about 7 to 16 carbonatoms, a non-terminal alkenyl of about 7 to 22 carbon atoms, or/&branched-chain alkyl of from about 13 to 22 carbon atoms, and X is —OH,—COOCH₃, —COOC₂H₅, —OCOCH₃, —SOCH₃, —P(CH₃)₂O, COOC₂H₄OC₄H₄OH,—COOCH(CHOH)₄CH₃OH, —COOCH₂CHOHCH₃, COOCH₂CH(OR″)CH₂OR″,—(OCH₂CH₂)_(m)OH, —COOR′, or —CONR₁₂, where R′ is H, —CR₃, —C₂H₅, —C₂H₇or —C₂H₄OH; R″ is —H, or a non-terminal alkenyl of about 7 to 22 carbonatoms; and m is 2-6; provided that when R″ is an alkenyl and X is —OH orCOOH, at least one double bond is in the cis-configuration.

5.2.3.1.2 Solubility Enhancers

[0116] Another way to increase the transdermal delivery of a drug is touse chemical solubility enhancers that increase the drug solubility inits vehicle. This can be achieved either through changing drug-vehicleinteraction by introducing different excipients, or through changingdrug crystallinity (Flynn and Weiner, 1993).

[0117] Solubility enhancers include water diols, such as propyleneglycol and glycerol; mono-alcohols, such as ethanol, propanol, andhigher alcohols; DMSO; dimethylformamide; N,N-dimethylacetamide;2-pyrrolidone; N-(2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone,1-dodecylazacycloheptan-2-one and othern-substituted-alkyl-azacycloalkyl-2-ones.

5.2.3.1.3 Combinations of Enhancers (Binary Systems)

[0118] U.S. Pat. No. 4,537,776 to Cooper contains a summary ofinformation detailing the use of certain binary systems for penetrationenhancement. European Patent Application 43,738, also describes the useof selected diols as solvents along with a broad category ofcell-envelope disordering compounds for delivery of lipophilicpharmacologically-active compounds. A binary system for enhancingmetaclopramide penetration is disclosed in UK Patent Application GB2,153,223 A, consisting of a monovalent alcohol ester of a C8-32aliphatic monocarboxylic acid (unsaturated and/or branched if C18-32) ora C6-24 aliphatic monoalcohol (unsaturated and/or branched if C14-24)and an N-cyclic compound such as 2-pyrrolidone or N-methylpyrrolidone.

[0119] Combinations of enhancers consisting of diethylene glycolmonoethyl or monomethyl ether with propylene glycol monolaurate andmethyl laurate are disclosed in U.S. Pat. No. 4,973,468 for enhancingthe transdermal delivery of steroids such as progestogens and estrogens.A dual enhancer consisting of glycerol monolaurate and ethanol for thetransdermal delivery of drugs is described in U.S. Pat. No. 4,820,720.U.S. Pat. No. 5,006,342 lists numerous enhancers for transdermal drugadministration consisting of fatty acid esters or fatty alcohol ethersof C₂ to C₄ alkanediols, where each fatty acid/alcohol portion of theester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970discloses penetration-enhancing compositions for topical applicationincluding an active permeant contained in a penetration-enhancingvehicle containing specified amounts of one or more cell-envelopedisordering compounds such as oleic acid, oleyl alcohol, and glycerolesters of oleic acid; a C₂ or C₃ alkanol and an inert diluent such aswater.

[0120] Other chemical enhancers, not necessarily associated with binarysystems, include dimethylsulfoxide (DMSO) or aqueous solutions of DMSOsuch as those described in U.S. Pat. No. 3,551,554 to Herschler; U.S.Pat. No. 3,711,602 to Herschler and U.S. Pat. No. 3,711,606 toHerschler, and the azones (n-substituted-alkyl-azacycloalkyl-2-ones)such as noted in U.S. Pat. No, 4,557,943 to Cooper.

[0121] In PCT/US96/12244 by Massachusetts Institute of Technology,passive experiments with polyethylene glycol 200 dilaurate (PEG),isopropyl myristate (IM), and glycerol trioleate (GT) result incorticosterone flux enhancement values of only 2, 5, and 0.8 relative tothe passive flux from PBS alone. However, 50% ethanol and LA/ethanolsignificantly increase corticosterone passive fluxes by factors of 46and 900.

[0122] Some chemical enhancer systems may possess negative side effectssuch as toxicity and skin irritations. U.S. Pat. No. 4,855,298 disclosescompositions for reducing skin irritation caused by chemicalenhancer-containing compositions having skin irritation properties withan amount of glycerin sufficient to provide an anti-irritating effect.The present invention enables testing of the effects of a large numberof enhancers on tissue barrier transport, such as transdermal transport,of a compound, pharmaceutical, or other component.

5.2.3.2 Mechanical Enhancers For Transdermal Delivery

[0123] For convenience, mechanical enhancers are defined as includingalmost any extraneous enhancer, such as ultrasound, mechanical orosmotic pressure, electric fields (electroporation or iontophoresis) ormagnetic fields.

[0124] There have been numerous reports on the use of ultrasound(typically in the range of 20 kHz to 10 MHz in frequency) to enhancetransdermal delivery. Ultrasound has been applied alone and incombination with other chemical and/or mechanical enhancers. Forexample, as reported in PCT/US96/12244 by Massachusetts Institute ofTechnology, therapeutic ultrasound (1 MHz, 1.4 W/cm²) and the chemicalenhancers utilized together produce corticosterone fluxes from PBS, PEG,IM, and GT that are greater than the passive fluxes from the sameenhancers by factors of between 1.3 and 5.0. Ultrasound combined with50% ethanol produces a 2-fold increase in corticosterone transport abovethe passive case, but increase by 14-fold the transport from LA/Ethanol,yielding a flux of 0.16 mg/cm²/hr, 13,000-fold greater than that fromPBS alone.

[0125] Pressure gradients can also be used to enhance movement of fluidsacross the skin. Pressure can be applied by a vacuum or a positivepressure device. Alternatively, osmotic pressure may be used to drivetransdermal transport.

[0126] Similarly, application an of electric current has been shown toenhance transdermal drug transport and blood analyte extraction. Suchelectric current enhances transport by different mechanisms. Forexample, application of an electric field provides a driving force forthe transport of charged molecules across the skin and second, ionicmotion due to application of electric fields may induce convective flowsacross the skin, referred to as electro-osmosis. This mechanism isbelieved to play a dominant role in transdermal transport of neutralmolecules during iontophoresis. Iontophoresis involves the applicationof an electrical current, preferably DC, or AC, at a current density ofgreater than zero up to about 1 mA/cm². Enhancement of skin permeabilityusing electric current to achieve transdermal extraction of glucose, wasreported by Tamada, et al., Proceed. Intern. Symp. Control. Rel. Bioact.Mater. 22, 129-130 (1995).

[0127] Application of magnetic fields to the skin pretreated or incombination with other permeation enhancers can be used to transportmagnetically active species across the skin. For example, polymermicrospheres loaded with magnetic particles could be transported acrossthe skin.

[0128] 5.2.4 Adhesives

[0129] Some devices for delivery of an active component or drug across atissue barrier, and in particular transdermal delivery devices such astransdermal patches, typically include an adhesive. The adhesive oftenforms the matrix in which the active component or drug is dissolved ordispersed and, of course, is meant to keep the device in intimatecontact with the tissue, such as skin. Compatibility of the activecomponent or drug with an adhesive is influenced by its solubility inthat adhesive. Any supersaturated conditions produced in storage or inuse are generally very stable against precipitation of the activecomponent or drug within the adhesive matrix. A high solubility isdesired in the adhesive to increase the driving force for permeationthrough the tissue and to improve the stability of the device.

[0130] Several classes of adhesive are used, each of which contain manypossible forms of adhesives. These classes include polyisobutylene,silicone, and acrylic adhesives. Acrylic adhesives are available in manyderivatized forms. Thus, it is often a very difficult problem to selectwhich adhesive might be best to use with any particular drug andenhancer. Typically, all ingredients to be in the device are dissolvedin a solvent and cast or coated onto a plastic backing material.Evaporation of the solvent leaves a drug-containing adhesive film. Thepresent invention enables rapid and efficient testing of the effects ofvarious types and amounts of adhesives in a sample composition orformulation.

[0131] 5.2.5 Solvents

[0132] Solvents for the active component, carrier, or adhesive areselected based on biocompatibility as well as the solubility of thematerial to be dissolved, and where appropriate, interaction with theactive component or agent to be delivered. For example, the ease withwhich the active component or agent is dissolved in the solvent and thelack of detrimental effects of the solvent on the active component oragent to be delivered are factors to consider in selecting the solvent.Aqueous solvents can be used to make matrices formed of water solublepolymers. Organic solvents will typically be used to dissolvehydrophobic and some hydrophilic polymers. Preferred organic solventsare volatile or have a relatively low boiling point or can be removedunder vacuum and which are acceptable for administration to humans intrace amounts, such as methylene chloride. Other solvents, such as ethylacetate, ethanol, methanol, dimethyl formamide (DMF), acetone,acetonitrile, tetrahydrofuran (THF), acetic acid, dimethyl sulfoxide(DMSO) and chloroform, and combinations thereof, also may be utilized.Preferred solvents are those rated as class 3 residual solvents by theFood and Drug Administration, as published in the Federal Register vol.62, number 85, pp. 24301-24309 (May 1997). Solvents for drugs willtypically be distilled water, buffered saline, Lactated Ringer's or someother pharmaceutically acceptable carrier.

[0133] 5.3 Sample Preparation and Screening Methods

[0134] The high throughput screening methods of the present inventionidentify, for example, 1) optimal compositions or formulationscomprising one or more active components and one or more inactivecomponents for achieving desired characteristics for such compositionsor formulations, 2) optimal adhesive/enhancer/excipient compositions forcompatibility with an active component or drug, 2) optimal activecomponent or drug/adhesive/enhancer/additive compositions for maximumdrug flux through stratum corneum, and 3) optimal active component ordrug/adhesive/enhancer/additive compositions to minimize cytotoxicity.

[0135] The basic requirements for sample preparation, processing, andscreening are a distribution mechanism and a testing, or screening,mechanism. The distribution mechanism adds components to separate siteson an array plate, such as into sample wells. Preferably, thedistribution mechanism is automated and controlled by computer softwareand can vary at least one addition variable, e.g., the identity of thecomponent(s) and/or the component concentration, more preferably, two ormore variables. For instance, filling or addition of a sample, such as apharmaceutical component and excipients (e.g., enhancers and adhesives)to a sample well involves material handling technologies and roboticswell known to those skilled in the art of pharmaceutical processmanufacturing. Of course, if desired, individual components can beplaced into the appropriate well in the array manually. This pick andplace technique is also known to those skilled in the art. A testingmechanism is preferably used to test each sample for one or moreproperties, such as drug concentration as a function of time.Preferably, the testing mechanism is automated and driven by a computer.

[0136] In one embodiment, the system further comprises a processingmechanism to process the samples after component addition. For example,after component addition to the sample well but prior to assembly of theapparatus and in particular placement of the tissue specimen over thesample well, the samples can be processed by stirring, milling,filtering, centrifuging, emulsifying, or solvent removal (e.g.,lyophilizing) and reconstituting, etc. by methods and devices well knownin the art. Preferably the samples are processed automatically andconcurrently.

[0137] As mentioned supra, a preferred method of using the tissuebarrier transfer device of FIG. 1 entails determining, directly orindirectly, the presence, absence or concentration of components (e.g.pharmaceuticals) that diffuse through tissue 120 into reservoir 132 ofthe array. Such measurements may be performed by a variety of meansknown to those skilled in the art. For example, any know spectroscopictechnique can be used to determine presence, absence or concentration ofa component-in-common. Suitable measurement techniques include, but arenot limited to include spectroscopy, infrared spectroscopy, nearinfrared spectroscopy, Raman spectroscopy, NMR, X-ray diffraction,neutron diffraction, powder X-ray diffraction, radiolabeling, andradioactivity.

[0138] In one exemplary embodiment, and not by way of limitation, thepassive permeabilities of active components (e.g. a drug) through humanskin can be measured using trace quantities of radiolabelled activecomponent or drug. According to known methods, radiolabelled compoundsor drugs are rotary evaporated in order to remove any solvent in whichthey are shipped and any tritium which had reverse exchanged into it.The radiolabelled compounds or drugs are then redissolved in variouscomposition formulations, including enhancers, carriers, additives,adhesives, and/or other excipients as described infra, to a typicalconcentration of 1 μChi/ml, and added to the sample wells, such assample wells 116 of array 112 in FIG. 1. Passive permeation experimentsare then performed. The reservoir compartments, such as reservoirs 132of FIG. 1., preferably contain, for example, pH 7.4 phosphate buffersaline (PBS, phosphate concentration=0.01 M, NaCl concentration=0.137 M)(Sigma Chemical Co.). Other receiver solutions may be used and are knownto those skilled in the art. The concentrations of radiolabelledcomponent or drug in the sample and reservoir compartments are measuredusing a scintillation counter (e.g., model 2000 CA, PackardInstruments). Duplicate formulations may be used in some of the samplesand/or repeated experiments may be performed to optimize reliability ofmeasurements.

[0139] The permeability values can be calculated under steady-stateconditions from the relationship P=(dN_(r)/dt)/(AC_(d)) where A is thesurface area of the tissue accessible to a sample, C_(d) is thecomponent or drug concentration in the sample, and N_(r) is thecumulative amount of component or drug which has permeated into thereceptor reservoir. Inter-subject variability of the human skinpermeability of 40%, is reported by Williams, et al., Int. J. Pharm. 86,69-77 (1992). The passive permeability enhancements, E_(p), iscalculated relative to the passive permeability from PBS according toEq. (1): $\begin{matrix}{E_{p} = \frac{P({enhancer})}{P({PBS})}} & (1)\end{matrix}$

[0140] where P(enhancer) is the drug permeability from a given enhancer,and P(PBS) is the drug permeability from PBS. The fluxes from saturatedsolutions, J^(sat), are calculated from J^(sat)=PC^(sat), where C^(sat)is the drug solubility in the formulation. Flux enhancements, E_(J), arecalculated using Eq. (2), $\begin{matrix}{E_{J} = \frac{\quad^{it}({enhancer})}{J^{sat}({PBS})}} & (2)\end{matrix}$

[0141] where J^(sat) (enhancer) and J^(sat) (PBS) are the drug fluxesfrom saturated solutions of enhancer and PBS, respectively.

[0142] 5.4 Correction or Repair of Microdefects in Skin Tissue Samples

[0143] The present invention includes a methods for repairing and/orcorrecting for microscopic defects on tissue specimens, such as skin.For example, apparatus or a diffusion cell used for study of transdermaldelivery of active components (e.g., pharmaceuticals or drugs) requireskin samples that are free of defect that might act as diffusional fasttransport paths. Such defects can be of several types with sizes rangingfrom millimeters to tens of microns. Physical tears and hair folliclesare just two types of defects that may compromise the interpretation oftransport or diffusion data. Inhomogeneous tissue segments, i.e.segments with an abnormal amount of defects, will lead to inaccurate andmisleading diffusion measurements, particularly when using relativelysmall tissue samples as in the present invention. Rapid identificationof defect locations on the surface of a given tissue sample may beachieved by image analysis, preferably by high-speed micro inspection ofeach tissue segment using video microscopy or photomicrography.

[0144] According to a preferred embodiment of the invention, diffusiondata related to inhomogeneous tissue segments may be discarded to avoidinaccurate measurements. Alternatively, if the effect of defects in atissue segment can be characterized and/or quantified, associateddiffusion measurements can be mathematically adjusted to account for thedefects.

[0145] In another embodiment of the invention, defects in a tissuespecimen are repaired by feeding the defect locations to an ink jetprinter that is instructed to print wax to cover these locations. Theprint pattern is devised so as to cover the entire area of the defectwith some possible overlap on to regions that are free of defects. Waxprint heads print molten wax that solidifies on impact with the tissue.The solid wax is water-resistant and acts like a seal to ensure that therepaired region does not contribute to the diffusional flux duringsubsequent testing. Droplet placement preferably is such that overlap issufficient to make a seal.

[0146] 5.5 Alternative Embodiment for Solid Source Samples (FIGS. 2A-2D)

[0147]FIGS. 2A-2D are schematic diagrams of an alternativehigh-throughput apparatus 200 and method for measuring tissue barriertransfer using a solid source sample. Apparatus 200 is similar toapparatus 100 of FIG. 1, except that apparatus 200 is designed fortesting solid source samples, such as compositions containing asemi-solid, such as an adhesive, a relatively flat transdermal patch, ora film-like sample. Substrate plate 214 is a dense plate, such as aplastic or glass plate, that supports an array 212 of samples 216. Eachsample includes a combination of components, including an activecomponent (e.g., a pharmaceutical) and at least one inactive component.Examples of suitable components are discussed above with respect to FIG.1.

[0148] A first step of the method involves creating an array 212 ofdifferent composition regions (i.e., samples 216) on dense substrate214. The array may be produced in any number of ways, but one simplemethod is to use combinatorial dispensing equipment to make solutions ofall the constituents in a convenient solvent. Suitable dispensingequipment and methods of formulating solutions or compositions arediscussed above and disclosed in U.S. patent application Ser. No.09/540,462, which is herein incorporated by reference in its entirety.

[0149] In a preferred embodiment, the formulated solutions are containedin the wells of a microtiter plate similar to substrate plate 114 (ofFIG. 1) that includes a sample array 112 of sample wells 116 andseparable dense bottom plate 214 rather than base 118. The solvent isthen evaporated and each of the samples in the wells is allowed to dryto leave a film at the bottom. This evaporation process mimics themanufacturing process used to make various tissue transfer devices, suchas transdermal patches. The upper plate may then be removed to yield thearray shown in FIG. 2A. The samples 216 can be any shape, and preferablyare generally round in shape as shown in FIG. 2A.

[0150] It should be noted that the plates of this format can be used toassess the stability of the compositions or formulations, such asdrug/adhesive/enhancer solutions, toward precipitation of the activecomponent, such as a pharmaceutical or drug. Optical examination of eachof the films will reveal if precipitation has occurred, since theprecipitates may cause increased light scattering when the sample isilluminated. Crystallization can also be optically detected by observingbirefringence of crystals (for compounds that are birefringent).Alternative means may be used when the film is already sufficientlyopaque to preclude the scattering method. One such method is secondharmonic generation (SHG) which easily detects the presence of crystalsin the film. It is also possible to use microfocus X-ray diffraction todetect the presence of crystals.

[0151] Referring to FIG. 2B, the next step of the present method is toprepare a tissue specimen 220 that is to be used in the study. Aspecimen 220, such as a specimen of stratum corneum, may beconventionally prepared or obtained as described above. It is mostconvenient, however, that the sample specimen 220 should be sufficientlylarge to cover whatever plate format is used for the study. For example,it should be sufficiently large to cover a 96 well microtiter plate.Thus, a separate tissue sample is prepared for each plate 214 of thestudy. The tissue is then placed on plate 214 so as to cover each of thesample regions, as shown in the FIG. 2B. Care is taken to insure that noair pockets are present under tissue 220. One approach is to lay tissue220 down on plate 214 starting at one edge and gently proceeding acrossthe surface of the plate. The air is expelled ahead of the tissue/platecontact line.

[0152] Referring to FIG. 2C, in one embodiment of the present invention,the region of tissue 220 above each sample region may now be physicallysectioned or isolated into segments 224 from neighboring regions toensure that lateral diffusion does not occur between adjacent samples.As described above, this can be done in any number of ways, such asmechanical scribing or cutting, laser cutting or crimping along cuts222.

[0153] Each of the tissue segments 224 on each plate 214 may now beimaged and characterized by video microscopy. Automated imagerecognition can be used to identify and record those tissue segmentsthat are damaged or otherwise inhomogeneous. As described above, damagedor inhomogeneous tissue segments 224 may be replaced, repaired orignored. Alternatively, data associated with damaged or inhomogeneoussegments 224 may be adjusted to account for the defects. Optionally,tissue 220 may be imaged and replaced or repaired prior to sectioning.In yet another alternative method, the tissue 220 is sectioned and/orimaged before placing tissue segments 224 over samples 216.

[0154] Referring to FIG. 2D, a next step in the present method is toplace a reservoir plate 230, similar to reservoir plate 130 of FIG. 1 oran open-bottomed titer plate, over the tissue segments 224 as shown.Reservoir plate 230 includes a number of hollow reservoirs 232. Whenplate 230 is secured in place, each reservoir 232 aligns over a sampleand tissue such that a tissue segment 224 separates each sample fromreservoir 232. Reservoir plate 230 secures to substrate plate 214 usingclamps, screws, fasteners, or any other suitable attachment means.Plates 230 and 214 preferably secure together with sufficient pressureso as to create a liquid tight seal around reservoirs 232. Eachreservoir is filled with a reservoir medium, preferably a liquid orsolution, such as a saline solution, to receive sample compounds thatdiffuse across tissue segments 224 to reservoir 232. In one embodiment,the reservoir medium is approximately 2% BSA solution in PBS.

[0155] Incubation of the apparatus 200 with automated periodic samplingand makeup of the reservoir 232 solution is used to assess thepermeability of the active component for all the samples of thecombinatorial study.

[0156] Although the embodiments of the invention described herein aredirected to movement of compounds across a tissue, the systems andmethods of the present invention are suitable for studying movement ofcompounds across any membrane or other barrier.

[0157] 5.6 Alternative Embodiment Using Indirect Measurement (FIG. 3)

[0158] In FIG. 3, another embodiment of the invention, apparatus 300relates to a method of high-throughput screening of active componentflux through a tissue specimen, such as the stratum corneum, recognizingthat such flux is determined, at least in part, by the permeability ofthe active component (such as a pharmaceutical or drug) within thetissue in the presence of an enhancer. The permeability is generallygoverned by at least two factors: the solubility of the active componentwithin the tissue (such as the stratum corneum) and the diffusivity ofthe active component within the tissue specimen. These two factors,solubility and diffusivity, are measured independently as a method ofindirectly assessing the flux through the tissue specimen.

[0159] Referring to FIG. 3, an array 312 of wells 316 containing samples(e.g. solutions 338) of different compositions of active components andinactive components (e.g., pharmaceutical/adhesive/enhancer/additive) isconstructed. Known amounts of tissue segments 340, e.g. stratum corneum,are added to each well. Alternatively, a tissue segment is placed on orover each well 316 (similar to the arrangement shown in FIGS. 1, 2C and2D) such that each segment is in contact with a sample solution 338. Therate at which a component (e.g., a drug, or pharmaceutical) is taken upinto the tissue sample may be measured by extracting the tissue 340 fromsimilarly prepared wells 316 at different times and measuring thepresence, absence, or concentration of the component. Measuring theconcentration after times sufficiently long so that the amount dissolvedis not changing with time can assess solubility, or the equilibriumconcentration of the component within the tissue 340. The product of therate and solubility is proportional to the permeability of thecomponent.

[0160] 5.7 Alternative Tissue Barrier Transfer Apparatus (FIGS. 4A-4C)

[0161] Referring to FIG. 4A, an alternative embodiment of the apparatusof FIG. 1 is diffusion cell 400. Diffusion cell 400 includes a sinkplate 410, a source plate 430, and a tissue specimen 420 disposedbetween sink plate 410 and source plate 430. Sink plate 410 includes asink well 412 for holding reservoir medium as described above withrespect to FIG. 1. Sink well 410 is shown as having a cylindrical shapewith an open end, however it may be rectangular, hexagonal, spherical,elliptical, or any other shape. Sink plate 410 includes at least oneaccess port 416 along an edge of sink well 412 that fluidly communicateswith sink well 412. Sink plate 410 also preferably includes a surfacefeature 414 configured to mate with source plate 430 and form a tightseal with tissue specimen 420.

[0162] In one preferred embodiment, tissue specimen 420 is skin tissue,but may be any tissue or membrane as described above with respect totissue specimen 120 of FIG. 1. Tissue specimen 420 is cut, formed orotherwise dimensioned to cover sink well 412 and surface feature 414.Tissue specimen 420 is placed such that it preferably does notcompletely cover access port 416.

[0163] Referring to FIG. 4C, source plate 430 includes a sourcereservoir, or well 432 that has open ends and aligns with sink well 412when source plate 430 is placed on tissue specimen 420. A passage 436also passes through source plate 430 and is approximately adjacent to,but not in communication with, source well 432. Passage 436 isconfigured to align with access port 416 to provide access to thereservoir medium in sink well 412 without removing source plate 430.

[0164] Referring again to FIG. 4A, source plate 430 also preferablyincludes a surface feature 434 that is configured and dimensioned tomate with surface feature 414 of sink plate 410 and form a seal withtissue specimen 420 around the perimeter of sink well 412 and sourcewell 432. For example, in one embodiment surface feature 414 is a convexring extending from the surface sink plate 420 around the open perimeterof sink well 412; and surface feature 434 is a concave ring formed insource plate 430 configured to mate with surface feature 414.

[0165] In another embodiment of the present invention, a numberdiffusion cells 400 are attached or formed together to create an arrayof diffusion cells similar to array 112 of FIG. 1.

[0166] Exemplary uses of the apparatus of FIGS. 4A-4C are the same asthose described above with respect to FIG. 1, except that access port416 and passage 436 allow addition or removal of reservoir medium fromsink well 412 without removing source plate 430 or tissue 420.Preferably, the reservoir medium used in diffusion cell 400 is a liquidor solution. In an alternative method of using diffusion cell 400, theplacement of reservoir medium and sample could be reversed as in FIG. 1;for example, reservoir medium could be placed above tissue specimen 420in source well 432 and sample could be held in sink well 412. In such anembodiment, sample may be added or removed through passage 436 andaccess port 416.

[0167] 5.8 Method For Filling or Adding Samples (FIGS. 5A & 5B)

[0168]FIGS. 5A and 5B show a schematic drawing of an apparatus 500 foruse in adding or filling a sample 530 into a sample well 522 in a samplearray, such as sample array 112 shown in FIG. 1, wherein the occurrenceof air pockets or bubbles between the sample 530 and a tissue 524 isavoided. In the sample array, the tissue 524 is located between a samplewell 522, which is located in a substrate plate, such as substrate plate114 shown in FIG. 1, and a reservoir 526, which is located in areservoir plate, such as reservoir plate 130 shown in FIG. 1. In thefilling method of the present invention, a feed canula 510, having asample feed source 514 and an air evacuation space 512, punctures a basemembrane 520 which covers one side a the sample well 522 to be filledwith sample 530.

[0169] Then, sample feed source 514 is extended into sample well 522until it is in contact with tissue 524. Sample 530 is then fed throughsample feed source 512, and as sample 530 begins to fill sample well522, air is forced out of sample well 522 through air evacuation space512 in feed canula 510. When the desired amount of sample 530 is filledinto sample well 522, sample feed source 512 and feed canula 510 arecompletely withdrawn from base membrane 520 and sample well 522.

[0170] In a preferred embodiment of the filling method of the presentinvention, while sample 530 is being fed into sample well 522, samplefeed source 514 retracts at a rate that is synchronized with the fillrate for sample 530 into sample well 522 such that at all times duringthe filling process, the outlet of sample feed source 514 is insideextruded sample 530 in sample well 522. When the desired amount ofsample 530 is filled into sample well 522, both sample feed source 512and feed canula 510 are completely withdrawn from base membrane 520 andsample well 522.

[0171] In a preferred embodiment, base membrane 520 is a rubbermembrane.

[0172] The filling method of the present invention can be performed byhand or using automated dispensing means, wherein sample wells in asample array are filled using automated dispensing equipment that iscapable of dispensing the same or different samples to multiple samplewells in one or more sample arrays in a fast, accurate, and controlledapproach.

[0173] Sample 530 dispensed in accordance with the filling method of thepresent invention is preferably a liquid source sample.

[0174] 5.9 Alternative Embodiments For Solid Source Samples (FIGS. 6-9)

[0175]FIG. 6 shows an exploded view, schematic diagram of a preferredembodiment of a high-throughput apparatus 600 for measuring tissuebarrier transport in an array of solid source samples 630 according tothe present invention. Apparatus 600 comprises a base plate 610supporting a spacer plate 620, an array of solid source samples 630, atissue specimen 640, a reservoir plate 650 having an array of donorreservoirs 654, and a clamping means, such as shoulder screws 660 withthreads 662.

[0176] Preferably, base plate 610 is made aluminum, and spacer plate 620and reservoir plate 650 are made of clear plastic or polycarbonate.

[0177] Base plate 610 has screw holes 612 which are drilled to mate withthreads 662 on shoulder screws 660, such that when screws 660 are fedthrew the apparatus into screw holes 612 and tightened, the apparatus isclamped together. When the apparatus is clamped together, a seal isformed between reservoir plate 650 and tissue specimen 640. There can beany number of screw holes 612 located around the edges of base plate610, but preferably, the number of screw holes 612 is at least 4, andmore preferably between 4 and 8. In a preferred embodiment, base plate610 further comprises an array of guide marks 614, which can be anyarray formation, such as 2×2, 4×4, 6×6, and 8×12, which are used to helpalign various components of apparatus 600 during assembly.

[0178] Screw holes 622 and screw holes 652 in spacer plate 620 andreservoir plate 650, respectively, are drilled to allow the neck andthreads 662 of shoulder screws 660 to smoothly pass through, but not thehead of shoulder screw 660 (as shown for shoulder screws 760 and 860 inFIGS. 7 and 8, respectively). There can be any number of screw holes 622and screw holes 652 located around the edges of spacer plate 620 andreservoir plate 650, respectively, but preferably, the number of screwholes 622 and screw holes 652 is at least 4, and more preferably between4 and 8. In a preferred embodiment, there is at least a screw hole ateach corner of both spacer plate 620 and reservoir plate 650.

[0179] In an alternative embodiment, apparatus 600 further comprises atop plate located above reservoir plate 650, which is made out of thesame material as base plate 610 (e.g., aluminum) and is either an openframe having screw holes matching screw holes 652 in reservoir plate 650or is a “solid” plate having the same screw holes and array ofreservoirs as screw holes 652 and donor reservoirs 654 on reservoirplate 650.

[0180] Apparatus 600 is assembled by first placing spacer plate 620 ontop of base plate 610 and aligning screw holes 622 in spacer plate 620with screw holes 612 in base plate 610. An array of solid source samples630 is created on spacer plate 620 in a pattern corresponding to thepattern of donor reservoirs 654 in reservoir plate 650, and guide marks614 on base plate 610 are used to ensure that each sample 630 is placedsuch that it aligns with a donor reservoir 654 in top plate 650. Thesize of samples 630 are commensurate with the size of donor reservoirs654.

[0181] Each sample 630 includes a combination of components, includingan active component (e.g., a pharmaceutical) and at least one inactivecomponent. Examples of suitable components are discussed above withrespect to FIG. 1.

[0182] A sheet of tissue specimen 640 is placed over the array ofsamples 630 in a manner which avoids formation of air pockets betweentissue specimen 640 and samples 630. Then, reservoir plate 650 having anarray of donor reservoirs 654 is placed over the skin such that screwholes 652 on top plate 650 align with the corresponding screw holes 622of spacer plate 620.

[0183] The resulting assembled apparatus 600 is then clamped together bysliding shoulder screws 660 with threads 622 through aligned screw holes652 of assembled apparatus 600, and each shoulder screw 660 is tightenedso as to form a seal between reservoir plate 650 and tissue specimen640. Preferably, a shoulder screw 660 should be used in at least each ofthe four corners of the assembled apparatus 600.

[0184] A reservoir medium is added to donor reservoirs 654 of assembledapparatus 600, and at an appropriate time or various time intervals,specimens are withdrawn from donor reservoirs 654 and used to measurethe transfer or flux of components, such as active components andcomponents-in-common, in samples 630 across tissue specimen 640. Ifmultiple specimens are taken, after a volume of specimen is removed froma donor reservoir 654, an equal volume of reservoir medium is added tothe same donor reservoir 654.

[0185] The size of donor reservoirs 654 is about 1 mm to about 50 mm,more preferably about 2 mm to about 10 mm, and most preferably about 3mm to about 7 mm.

[0186]FIG. 7 shows a compressed view, schematic diagram of ahigh-throughput apparatus 700 for measuring tissue barrier transport inan array of solid source samples according to the present invention.Apparatus 700 is the similar to apparatus 600, except the array of donorreservoirs 754 is an 8×12 array for a total of 96, wherein eachreservoir is no more than 6 mm in diameter. Apparatus 700 comprises abase plate 710 supporting a spacer plate 720, an array of solid sourcesamples (such as samples 630 shown in FIG. 6), a tissue specimen (suchas tissue specimen 640 shown in FIG. 6), a reservoir plate 750 having anarray of donor reservoirs 754, and a clamping means, such as shoulderscrews 760.

[0187]FIG. 8 shows a compressed view, schematic diagram of ahigh-throughput apparatus 800 for measuring tissue barrier transport inan array of solid source samples according to the present invention.Apparatus 800 is the similar to apparatus 600 and apparatus 700, exceptthe array of donor reservoirs 854 is a 16×24 array for a total of 384donor reservoirs 854 wherein each reservoir is no more than 3 mm indiameter. Apparatus 800 comprises a base plate 810 supporting a spacerplate 820, an array of solid source samples (such as samples 630 shownin FIG. 6), a tissue specimen (such as tissue specimen 640 shown in FIG.6), a reservoir plate 850 having an array of donor reservoirs 854, and aclamping means, such as shoulder screws 860.

[0188] Variations to the apparatuses of FIGS. 7 & 8 are the same asthose described for FIG. 6, and other variations to and exemplary usesof the apparatuses of FIGS. 6-8 are the same as those described abovewith respect to FIG. 1, where applicable.

[0189]FIG. 9 is an exploded view of a another transdermal assayapparatus according to another embodiment of the invention. Thetransdermal assay apparatus 900 is similar to the apparatuses 600, 700,and 800 described above, as the apparatus 900 is also used for measuringthe transport of a sample through a tissue barrier or specimen. Thesample is preferably a solid source sample, but alternatively may be anyother suitable sample.

[0190] Transdermal assay apparatus 900 comprises first and secondmembers 902 and 904, respectively. The first and second members arepreferably made from stainless steel, coated appropriately to obtain thedesired chemical resistance. These members could also be made from aclear plastic, such as polycarbonate, other plastics, or glass. Thefirst member 902 has opposing sides 905 and 922. These opposing sidesare preferably substantially planar surfaces that are substantiallyparallel to one another. One side 905 includes one or more samplesurfaces 906 configured to receive one or more samples 908 thereon. Thesample surfaces 906 are preferably circular and arranged in an array, asshown.

[0191] The second member 904 also has opposing sides 910 and 914 thatare also preferably substantially planar surfaces that are substantiallyparallel to one another. The second member 904 defines one or morereservoirs 912 therein. The size of each reservoir 912 is similar tothat described above in relation to FIGS. 7-8. Each reservoir has anopening 934 on the side 910 of the second member 904. In a preferredembodiment, each opening 934 is substantially the same size as eachsample surface 906.

[0192] In use, each sample surface 906 is configured to substantiallyalign with a corresponding opening 934 in a direction substantiallyperpendicular to the sample surface 906. Accordingly, in use, eachsample 908 of an array of samples is configured to substantially alignwith a corresponding opening 934 of an array of openings. In oneembodiment, the reservoirs 912 extend entirely through the second member904 from the surface 910 to the opposing side 914, in a similar mannerto that described above.

[0193] The transdermal assay apparatus 900 also preferably comprises athird member 916. The third member 916 is preferably made from aluminum,but could also be made from a number of other materials, such as steels,brass, plastics, ceramics or the like, that can be manufactured into apart that provides the desired geometry and dimensional stability. Thethird member 916 has opposing sides 918 and 920 that are also preferablysubstantially planar surfaces that are substantially parallel to oneanother. The third member 916 preferably includes a cavity 924 thereinfor receiving a magnet 926. In an alternative embodiment, the magnet 926is permanently formed in the third member 916, bonded to the thirdmember, or the third member 916 itself is the magnet 926, i.e., thethird member is made from a magnetic material. The magnet 926 may be apermanent magnet, electromagnet, or the like.

[0194] The magnet 926 forms one portion of a magnetic clamp used toclamp first 902, second 904, and third members 916 together. The otherportion of the magnetic clamp preferably comprises one or more inserts928 embedded in the second member 904. In a preferred embodiment, theseinserts 928 are made from a ferrous material that is attracted to themagnet 926. In an alternative embodiment, the inserts 928 are magneticinserts that are attracted to the magnet 926, i.e., the magnetic insertsand the magnet 926 are arranged to have opposite polarities facing oneanother. These magnetic inserts are preferably permanent magnets, butalternatively may be electromagnets. In a preferred embodiment, thefirst and third members are made from a ferrous or magnetic material(such as 430 stainless steel). These magnetic members are then attractedto hold by the magnet in the third member to provide the desiredclamping force.

[0195] The third member also preferably includes at least one, butpreferably two or more, alignment posts 930 configured to mate withcomplementary alignment holes 932 in the first member 902. The alignmentposts are preferably made from a ferrous material to aid the attractionbetween the magnet 926 and the inserts 928, and to hold the magnet 926in place in the cavity 924.

[0196] It should be appreciated that in alternative embodiments themagnetic clamp may take on various other forms. For example, themagnetic clamp may comprise one or more magnets and/or ferrous insertsdisposed in, on, or near the first member 902, second member 904, and/orthird member 916. In addition, these members may be fully or partiallymade from ferrous and/or magnetic materials. In addition, the strengthof the magnet 926, or the gap between the magnet(s) and insert(s) ormagnetic material(s) is preferably chosen to provide the requiredclamping force between the members. In other words, the magnet 926 canbe replaced by a stronger or weaker magnet to alter the resultingclamping force and to optimize the sealing among the members of theapparatus 900.

[0197] In a preferred method of use, the magnet 926 is bonded to thethird member 916. A sample 908 is arranged on each sample surface 906 ofthe first member 902, such that each sample surface 906 will align witha corresponding opening 934. The size of the samples 908 arecommensurate with the size of each opening 934. Each sample 908 includesa combination of components, including an active component (e.g., apharmaceutical) and at least one inactive component. Examples ofsuitable components are discussed above with respect to FIG. 1.

[0198] The second member 902 is then placed onto the third member 916such that the alignment posts 930 mate with the alignment holes 932.This aligns the second member 902 in a predetermined position on thethird member 916. A sheet of tissue specimen 936 is placed over thesamples 908 in a manner which avoids formation of air pockets betweenthe tissue specimen 936 and the samples 908.

[0199] The second member 904 is then placed over the tissue specimen936, such that the tissue specimen 936 is exposed to each reservoir'sopening 934. A magnetic attraction between the magnet 926 and inserts928, preferably along the alignment posts 930, aligns the second member904 with the first member 902, such that each sample 908 is alignedunder a corresponding opening 934. This magnetic attraction between themagnet 926 and inserts 928 also creates a clamping force that clamps thefirst, second, and third members together, thereby clamping the tissuespecimen between each sample 908 and each opening 934.

[0200] A fluid medium is added to the reservoirs 912 of assembledapparatus 900, and at an appropriate time or various time intervals,specimens of the fluid medium are withdrawn from reservoirs 912 and usedto measure the transfer or flux of components, such as active componentsand components-in-common, in the samples 908 across the tissue specimen936. If multiple specimens are taken after a volume of specimen isremoved from a reservoir 912, an equal volume of fluid medium is addedto the same reservoir 912.

[0201]FIG. 10 is an exploded view of a yet another transdermal assayapparatus 1000 according to yet another embodiment of the invention. Thetransdermal assay apparatus 1000 is similar to the transdermal assayapparatus 900 (FIG. 9) described above. Unlike the transdermal assayapparatus 900 (FIG. 9), the transdermal assay apparatus 1000 includes asample plate and flexible magnet, as described below. In addition, thesample is preferably a solid source sample, but alternatively may be anyother suitable sample.

[0202] Transdermal assay apparatus 1000 comprises a first member 1010and a second member 1002. These members are preferably made fromstainless steel, coated appropriately to obtain the desired chemicalresistance. These members could also be made from a clear plastic, suchas polycarbonate, other plastics, or glass. These members alsopreferably have opposing planar surfaces and are substantially parallelto one another. The second member 1002 defines one or more reservoirs1014 therein, each having an opening, as described above in relation toFIG. 9.

[0203] In use, a flexible magnet 1008 is disposed between the first andsecond plates. A sample array, preferably disposed on a sample plate1006 is disposed between the flexible magnet 1008 and the second plate.Similarly, a tissue sample 1004 is disposed between the sample plate1006 and the second plate 1002.

[0204] The sample plate 1006 includes one or more sample surfaces 1012configured to receive one or more samples thereon. The sample surfaces1012 are preferably circular and arranged in an array, as shown. In use,each sample surface 1012 is configured to substantially align with acorresponding opening of each reservoir 1014 in a directionsubstantially perpendicular to the sample surface 1012. Accordingly, inuse, each sample of an array of samples is configured to substantiallyalign with a corresponding opening of an array of openings. In oneembodiment, the reservoirs 1014 extend entirely through the secondmember 1002, in a similar manner to that described above.

[0205] The flexible magnet 1008 forms one portion of a magnetic clampused to clamp the first 1010 and second 1002 members together. Thesecond plate 1002 is preferably made from a ferrous material that isattracted to the flexible magnet 1008. In an alternative embodiment,ferrous inserts are placed in the second member 1002.

[0206] The transdermal assay apparatus 1000 is used as described above.

6. EXAMPLE

[0207] The following Example further illustrates the method and arraysof the present invention. It is to be understood that the presentinvention is not limited to the specific details of the Example providedbelow.

Example 1 Nicotine Permeation Across Human Cadaver Skin

[0208] Human cadaver skin epidermis was prepared by first separatingskin from the underlying fat and then separating the epidermis by heattreatment at 60° C. for 90 seconds using standard techniques.

[0209] A NICODERM CQ® brand nicotine Step 1 (21 mg/24 hours) transdermalpatch (sold by GlaxoSmithKline, Research Triangle Park, N.C. USA) waspunched into {fraction (5/16)}″ diameter circles, keeping the backingand release liners on the resulting punched samples until such weredeposited in the test apparatus.

[0210] An apparatus as described in FIG. 6 was assembled, wherein eachplate in the apparatus was a rectangular shape having dimensions of5.030″ (127.76 mm) by 3.365″ (85.48 mm). The apparatus was assembled byfirst placing a {fraction (1/8)}″ (3.175 mm) thick clear polycarbonatespacer plate 620 on top of an aluminum base plate 610 and aligning screwholes 622 in the spacer plate with screw holes 612 in the base plate.Thereafter, a 4×4 sample array was created on spacer plate 620, asdescribed in Table 2 below: TABLE 2 4 × 4 Test Array 1 2 3 4 A samplesample sample sample* B sample sample empty (control) sample C empty(control) sample sample sample D sample* sample empty (control) sample

[0211] Punched samples were placed on spacer plate 620 in the 4×4 arrayof Table 2 one at a time, and the location of each array sample wasselected using guide marks 614 on base plate 610 to ensure that eacharray sample was placed such that it aligned with a donor reservoir 654in reservoir plate 650. There were 96, 0.020″ (0.508 mm) deep, guidemarks 614 on base plate 610, arranged in a 12×8 array which was located11.24 mm in from the edges of the long sides of base plate 610, and14.38 mm in from the edges of its short sides. At the time of placement,the release liner on each sample was removed to expose the drugreservoir/adhesive of the sample.

[0212] In array samples A4 and D1, the drug reservoir of the samplepatch came off of the backing with the release liner. Array locationsB3, C1, and D3 were controls without any samples in order to determinethe potential impact of lateral diffusion on transdermal transportmeasurements with this apparatus.

[0213] Once all the samples were placed in the array, the piece ofheat-stripped human cadaver skin, the size of which was larger than thearray of samples, was gently and slowly placed over the samples so as toavoid any air pockets between the skin and the samples. The skin wasoriented with the stratum corneum next to the samples. Then, a {fraction(1/4)}″ (6.35 mm) thick clear polycarbonate reservoir plate 650 havingan 4×4 array of {fraction (1/4)}″ (6.35 mm) diameter donor reservoirs654 was placed over the skin such that all of screw holes 652 onreservoir plate 650 were aligned with the corresponding screw holes 622of spacer plate 620.

[0214] The resulting assembled apparatus 600 was clamped together bysliding a shoulder screw 660 with threads 622 through aligned screwholes 652 at each of the four corners of the assembled apparatus, andtightening each shoulder screw 660 so as to form a seal betweenreservoir plate 650 and the skin. The screw holes 612 on base plate 610had a 10-24 tap, ranging between 0.250″ (6.35 mm) and 0.188 (4.775 mm),which gripped threads 662 of screws 660 as the screws were tightened,thereby clamping the apparatus together.

[0215] 75 ml of Dulbecco's Phosphate Buffered Saline (PBS) was added toeach donor reservoir in the array as the reservoir medium.

[0216] After 2 hours, a 50 ml test aliquot of reservoir medium wasremoved from each donor reservoir 654, and at that time, an additional50 ml of PBS was added into each donor reservoir 654. Each of the 2-hourtest aliquots was placed in an HPLC vial and diluted to 500 ml byaddition of 450 ml of 50:50 (v/v) 50 mM potassium phosphate (adjusted topH 3.0 with phosphoric acid) and acetonitrile.

[0217] The foregoing process was repeated at 3 hours, 4 hours, and 5hours. At the end of the sampling phase of the experiment, each donorreservoir 654 in the array resulted in four (4) 50 ml test aliquots thatwere diluted as set forth above, except that the test aliquot taken at 3hours for the B3 donor reservoir was diluted to 950 ml rather than 500ml.

[0218] The nicotine content in each test aliquot was then determined byHPLC analysis.

[0219] The components of the HPLC system used to analyze the testaliquots were a Waters 2790 Separations Module, a Waters PhotodiodeArray Detector Model 996, and Waters Millennium 32 v3.2 ChromatographySoftware (Waters Corp., Milford Mass.).

[0220] The HPLC analysis was performed using a Platinum EPS C18 column(Alltech Associates, Muskegan, Mich.) with dimensions of 250 mm×4.6 mmand a 5 mm particle size. The mobile phase was 50:50 (v/v) 50 mMpotassium phosphate (adjusted to pH 3.0 with phosphoric acid):acetonitrile, with a flow rate of 1.0 ml/minute. Detection was performedby measuring UV absorbance at a wavelength of 260 nm. The run time was 4minutes. Injection volume was 10 ml. Column temperature was ambient.

[0221] Quantification of nicotine content in each test aliquot wasperformed by comparison to a calibration curve generated using a set ofnicotine standards (Sigma). Nicotine quantitation was shown to be linearover a range of 1-100 mg/ml. Potential chromatographic interference withthis method of other components (e.g., fat, protein) in the skin wasruled out by direct analysis.

[0222] The results of the HPLC analysis are set forth in Tables 3 and 4below: TABLE 3 Nicotine Concentration Of Diluted Test AliquotsConcentration (mg/ml) 2 hour 3 hour 4 hour 5 hour A1 39.7 37.7 33.0 33.5A2 51.2 26.7 42.5 42.4 A3 49.3 40.7 30.5 33.2 A4 1.1 1.4 1.6 1.8 B1 11.316.8 17.1 16.7 B2 25.4 33.0 36.6 30.1 B3 2.2 2.5 4.8 6.8 B4 28.1 36.133.3 32.7 C1 1.5 3.2 2.7 2.6 C2 37.5 37.0 33.4 29.1 C3 10.1 12.9 13.115.2 C4 27.2 32.7 38.0 34.2 D1 1.0 1.2 1.4 1.6 D2 15.4 25.2 28.8 29.5 D32.6 4.0 4.2 4.7 D4 37.4 36.1 39.3 31.5

[0223] TABLE 4 Nicotine Concentration Of Original Test AliquotsConcentration (mg/ml) 2 hour 3 hour 4 hour 5 hour A1 397.0 377.0 330.0335.0 A2 512.0 267.0 425.0 424.0 A3 493.0 407.0 305.0 332.0 A4 11.0 14.016.0 18.0 B1 113.0 168.0 171.0 167.0 B2 254.0 330.0 366.0 301.0 B3 22.025.0 48.0 68.0 B4 281.0 361.0 333.0 327.0 C1 15.0 32.0 27.0 26.0 C2375.0 370.0 334.0 291.0 C3 101.0 129.0 131.0 152.0 C4 272.0 327.0 380.0342.0 D1 10.0 12.0 14.0 16.0 D2 154.0 252.0 288.0 295.0 D3 26.0 40.042.0 47.0 D4 374.0 361.0 393.0 315.0

[0224] The accumulation of nicotine in each donor reservoir wascalculate according to the following equations, Eq. (3)-(6):

Ac _(2hr)(mg)=[C ₂]×0.075 ml  (3)

AC _(3hr)(mg)=[C ₃]×0.075 ml+([C ₂]×0.05 ml)  (4)

AC _(4hr)(mg)=[C ₄]×0.075 ml+(([C ₂ ]+[C ₃])×0.05 ml)  (5)

AC _(5hr)(mg)=[C ₅]×0.075 ml+(([C ₂ ]+[C ₃ ]+[C ₄])×0.05 ml)  (6)

[0225] The results of the nicotine accumulation calculation for eachdonor reservoir in the array are set forth in Tables 6A and 6B below:TABLE 6A Nicotine Accumulation For Reservoirs A1 to B4 NicotineAccumulation (mg) A1 A2 A3 A4 B1 B2 B3 B4 0 hr 0 0 0 0 0 0 0 0 2 hr29.775 38.400 36.975 0.825 8.475 19.050 1.650 21.075 3 hr 48.125 45.62555.175 1.600 18.250 37.450 4.663 41.125 4 hr 63.450 70.825 67.875 2.45026.875 56.650 7.075 57.075 5 hr 80.325 92.000 85.150 3.400 35.125 70.07510.975 73.275

[0226] TABLE 6B Nicotine Accumulation For Reservoirs C1 to D4 NicotineAccumulation (mg) C1 C2 C3 C4 D1 D2 D3 D4 0 hr 0 0 0 0 0 0 0 0 2 hr1.125 28.125 7.575 20.400 0.750 11.550 1.950 28.05 3 hr 3.150 46.50014.725 38.125 1.400 26.600 4.300 45.775 4 hr 4.375 62.300 21.325 58.4502.150 41.900 6.450 66.225 5 hr 5.650 75.775 29.450 74.600 3.000 56.8258.925 80.025

[0227] As shown in the foregoing results, the active component,nicotine, was detected in donor reservoirs, and thus nicotine crossedthe skin barrier. These results also indicate that the detection ormeasuring method used was sufficiently sensitive to detect thetransported nicotine. There clearly was transdermal movement of theactive component, nicotine, and most of the samples demonstrated similarrates of transport.

[0228] In addition, substantially lower amounts of nicotine weredetected in donor reservoirs that were not located over samples,demonstrating that lateral diffusion of nicotine to adjacent “wells” wassufficiently slower than direct transdermal movement. Thus, thisexperiment clearly demonstrates the ability of this apparatus to measuretransport of a component across a tissue barrier.

[0229] The foregoing descriptions of specific embodiments of the presentinvention are presented for purposes of illustration and description.They are not intended to be exhaustive or to limit the invention to theprecise forms disclosed, obviously many modifications and variations arepossible in view of the above teachings. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical applications, to thereby enable others skilled in the artto best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated.Furthermore, the order of steps in the method are not necessarilyintended to occur in the sequence laid out. It is intended that thescope of the invention be defined by the following claims and theirequivalents. In addition, a number of references have been cited herein,all of which are hereby incorporated herein by reference.

We claim:
 1. A transdermal assay apparatus, comprising: a) a first lowermember having a sample surface configured to receive an array of samplesand having an array of samples provided on said sample surface; b) atissue specimen that overlays said array of samples; c) a second uppermember having an array of reservoirs each having an opening on thesurface of said second member, wherein said sample surface array andsaid reservoir array are substantially aligned with one another when inuse; wherein said array of samples provided on said first member has anactive component that transfers across said tissue specimen into saidarray of reservoirs of said second member.
 2. The transdermal assayapparatus of claim 1, wherein said sample surface and said opening areof substantially equal size.
 3. The transdermal assay apparatus of claim1, wherein said first member includes multiple sample surfaces and saidsecond member includes multiple reservoirs each having an opening on thesurface of said second member.
 4. The transdermal assay apparatus ofclaim 1, wherein in use said sample surface and said opening aresubstantially aligned with one another along a line substantiallyperpendicular with said sample surface.
 5. The transdermal assayapparatus of claim 1, wherein said reservoir is configured to receive atleast one fluid medium therein.
 6. The transdermal assay apparatus ofclaim 1, wherein said first and second members are made from a materialselected from a group consisting of: stainless steel, plastic,polycarbonate, glass, aluminum, brass, ceramic, and any combination ofthe aforementioned.
 7. The transdermal assay apparatus of claim 1,wherein said apparatus further comprises a liquid source sample.
 8. Thetransdermal assay apparatus of claim 1, wherein said tissue specimen isskin, lung, tracheal, nasal, placental, vaginal, rectal, colon, gut,stomach, bladder, corneal tissue, stratum corneum, living tissueexplants, animal tissue, human tissue, or engineered tissue-equivalents.9. The transdermal assay apparatus of claim 8, wherein the tissuespecimen is divided into a plurality of segments by mechanical cutting,scribing, laser cutting, scoring or crimping.
 10. The transdermal assayapparatus of claim 8, wherein the tissue specimen comprises skin tissue.11. The transdermal assay apparatus of claim 10, wherein the skin tissuecomprises epidermis or stratum corneum.
 12. The transdermal assayapparatus of claim 10, wherein the skin tissue is human skin tissue,animal skin tissue, or engineered skin tissue.
 13. The transdermal assayapparatus of claim 10, wherein the skin tissue comprises stratumcorneum.
 14. The transdermal assay apparatus of claim 10, wherein theskin tissue consists of stratum corneum.
 15. The transdermal assayapparatus of claim 1, further comprising a magnetic clamp configured toclamp said tissue specimen between said sample surface and said opening.16. The transdermal assay apparatus of claim 15, wherein said magneticclamp comprises: at least one magnet coupled to said first member; andat least one ferrous insert coupled to said second member.
 17. Thetransdermal assay apparatus of claim 15, wherein said magnetic clampcomprises: at least one magnet coupled to said first member; and atleast one magnetic insert coupled to said second member.
 18. Thetransdermal assay apparatus of claim 15, wherein said magnetic clampcomprises: at least one magnet coupled to said first member; and atleast a portion of said second member made from a ferrous material. 19.The transdermal assay apparatus of claim 15, wherein said magnetic clampcomprises: at least a portion of said first member made from a magneticmaterial; and at least a portion of said second member made from aferrous material.
 20. The transdermal assay apparatus of claim 15,wherein said magnetic clamp comprises: at least a portion of said firstmember made from a magnetic material; and at least a portion of saidsecond member made from a magnetic material.
 21. The transdermal assayapparatus of claim 15, wherein said magnetic clamp includes a permanentmagnet.
 22. The transdermal assay apparatus of claim 15, wherein saidmagnetic clamp includes an electromagnet.
 23. The transdermal assayapparatus of claim 15, wherein said magnetic clamp includes a magnethaving a strength that is selected based on the clamping force requiredbetween said first member and said second member.
 24. The transdermalassay apparatus of claim 15, further comprising a third memberconfigured to clamp said first member between said third member and saidsecond member.
 25. The transdermal assay apparatus of claim 24, whereinsaid magnetic clamp comprises: at least one magnet coupled to said thirdmember; and at least one ferrous insert coupled to said second member.26. The transdermal assay apparatus of claim 25, wherein said thirdmember comprises a cavity for receiving said at least one magnettherein.
 27. The transdermal assay apparatus of claim 24, wherein saidmagnetic clamp comprises: at least one magnet coupled to said thirdmember; and at least one magnetic insert coupled to said second member.28. The transdermal assay apparatus of claim 24, wherein said magneticclamp comprises: at least one magnet coupled to said third member; andat least a portion of said second member made from a ferrous material.29. The transdermal assay apparatus of claim 24, wherein said magneticclamp comprises: at least a portion of said third member made from amagnetic material; and at least a portion of said second member madefrom a ferrous material.
 30. The transdermal assay apparatus of claim24, wherein said magnetic clamp comprises: at least a portion of saidthird member made from a magnetic material; and at least a portion ofsaid second member made from a magnetic material.
 31. The transdermalassay apparatus of claim 24, wherein: said first member includes atleast one alignment hole; and said third member comprises at least onealignment post configured to mate with said at least one alignment hole.32. The transdermal assay apparatus of claim 24, wherein said thirdmember is made from a material selected from a group consisting of:aluminum, steel, brass, plastic, ceramic, and any combination of theaforementioned.
 33. The transdermal assay apparatus of claim 1, whereinsaid array of reservoirs contain a reservoir medium.
 34. The transdermalassay apparatus of claim 1, wherein said samples comprise acomponent-in-common and at least one additional component, wherein eachsample differs from at least one other sample with respect to at leastone of: (i) the identity of the additional components, (ii) the ratio ofthe component-in-common to the additional component, or (iii) thephysical state of the component-in-common.
 35. The transdermal assayapparatus of claim 34, wherein the component-in-common is apharmaceutical.
 36. The transdermal assay apparatus of claim 34, whereinthe component-in-common is a pharmaceutical, a dietary supplement, anutraceutical, or an alternative medicine.
 37. The transdermal assayapparatus of claim 34, wherein the additional component is an adhesive,an enhancer, an additive, a solvent, an excipient, or a combinationthereof.
 38. The transdermal assay apparatus of claim 37, wherein theadditional component is an adhesive.
 39. The transdermal assay apparatusof claim 38, wherein the adhesive is a polyisobutylene, a silicone, oran acrylic adhesive.
 40. The transdermal assay apparatus of claim 37,wherein the additional component is an enhancer.
 41. The transdermalassay apparatus of claim 40, wherein the enhancer is a chemicalenhancer, a lipid permeation enhancer, a solubility enhancer, or acombination of enhancers.